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Recent Advances in the Analysis of Steroid Hormones and Related Drugs

Recent Advances in the Analysis of Steroid Hormones and Related Drugs

ANALYTICAL SCIENCES MAY 2004, VOL. 20 767 2004 © The Society for Analytical Chemistry

Reviews Recent Advances in the Analysis of and Related Drugs

Sándor GÖRÖG

Gedeon Richter Ltd., P.O.B. 27, H-1475 Budapest, Hungary

The development during the last 15 years and the state-of-the-art in the analysis of bulk steroid drugs and hormone-like structures and pharmaceutical formulations made thereof are summarized. Other (sterols, acids, cardiac glycosides, vitamins D) as well as biological-clinical aspects and pharmacokinetic and metabolic studies are excluded from this review. The state-of-the-art is summarized based on comparisons of monographs in the latest editions of the European Pharmacopoeia, Pharmacopoeia and the Japanese Pharmacopoeia. This is followed by sections dealing with new developments in the methodology for the fields of spectroscopic and spectrophotometric, chromatographic, electrophoretic and hyphenated techniques as well electroanalytical methods. The review is terminated by two problem-oriented sections: examples on impurity and degradation profiling as well as enantiomeric analysis.

(Received January 14, 2004; Accepted February 2, 2004)

1 Introduction 767 4·3 Supercritical fluid chromatography (SFC) 2 Drugs in Pharmacopoeias 768 4·4 High-performance liquid chromatography 2·1 Assay of bulk drug materials (HPLC) and HPLC-MS 2·2 Related impurities test of bulk drug materials 5 Electrophoretic and Related Methods 776 2·3 Assay of steroid hormone formulations 5·1 Capillary electrophoresis (CE) 3 Spectroscopic and Spectrophotometric Methods 772 5·2 Micellar electrokinetic chromatography (MEKC) 3·1 Ultraviolet-visible spectrophotometry 5·3 Microemulsion electrokinetic chromatography 3·2 Fluorimetry (MEEKC) 3·3 Infrared and Raman spectroscopy 5·4 Capillary electrochromatography (CEC) and 3·4 Mass spectrometry CEC-MS 3·5 NMR spectroscopy 6 Electroanalytical Methods 777 3·6 Chemiluminometry 7 Selected Analytical Tasks 777 3·7 Circular dichroism (CD) spectropolarimetry 7·1 Impurity and degradation profiling of steroid 4 Chromatographic and Hyphenated Methods 774 drugs 4·1 Planar chromatography 7·2 Enantiomeric analysis 4·2 Gas chromatography (GC) and GC-MS 8 References 778

development in steroid hormone drug analysis during the last 15 1 Introduction years can be characterized by the facts that HPLC undoubtedly became the most important routinely used method and that the The aim of this paper is to give an overview of the advances importance of hyphenated chromatographic–spectroscopic during the last 15 years and of the state-of-the-art in the analysis techniques is increasing. New techniques, mainly capillary of steroid hormone drugs and related materials. Why just 15 electrophoresis (CE) and related techniques, such as micellar (or years? My first book on steroid hormone drug analysis was microemulsion) electrokinetic chromatography (MEKC, published in 19782 when this area was in transition state due to MEEKC), capillary electrochromatography (CEC), also attract the introduction and rapid spreading of new techniques, mainly wide interest in steroid analysis. Summarization of the results high-performance liquid chromatography (HPLC) and achieved with these methods will be the subject of this review. immunoassay methods. This was followed by another book in Only hormone drugs, their semi-synthetic analogues and 19833 dealing with wider areas (in addition to the analysis of hormone-like structures are considered. Other groups of hormone drugs other types of steroids and biological-clinical steroids (sterols, bile acids, cardiac glycosides, vitamins D, etc.) aspects). The third book, published in 19894 (just 15 years ago), are not within the scope of this review. Pharmaceutical- dealt mainly with pharmaceutical and industrial aspects. The industrial aspects are discussed, such as assay and impurity profiling of bulk drugs and drug formulations, estimating This paper is Part 56 in a series on “Analysis of Steroids”; for degradation profiles. Biological-clinical aspects, Part 55 see Ref. 1. pharmacokinetic and metabolic studies are also not within the E-mail: [email protected] scope of this review. These aspects are dealt with in a book of 768 ANALYTICAL SCIENCES MAY 2004, VOL. 20

Makin et al.5 Spectroscopic studies are not discussed either, pancuronium bromide6,8 and stanozolol6,7 with acetous unless these are part of impurity profiling and degradation perchloric acid. The group in ,7 after profiling studies. opening it with sodium hydroxide and the –O–SO2–ONa group in sodium sulfate8 after Na+ → H+ exchange, are also suitable for titrimetric determination. 2 Steroid Hormone Drugs in Pharmacopoeias Of the chromatographic methods other than HPLC, gas chromatography is only very seldom used for the assay of bulk Pharmacopoeias are naturally rather conservative: new steroid hormone drugs. The outdated and labor-consuming techniques appear in their monographs only if proved by many method of TLC separation and UV spectrophotometry after spot years of practice that the performance of these methods is elution is prescribed for the assay of , superior to that of the currently used methods. In spite of this phenylpropionate, hemisuccinate and limitation, the state-of-the-art of methodology and requirements .7 in official drug analysis is well reflected by monographs concerning the latest revisions of the principal pharmacopoeias. 2·2 Related impurities test of bulk drug materials The test for “Related substances”,6 also named 2·1 Assay of bulk drug materials “Chromatographic purity”,7 “Ordinary impurities”,7 “Related Table 1 shows a brief summarization of the assay and related steroids”7 or “Other steroids”,8 is the most important test to impurities tests in the monographs of bulk steroid hormones and characterize the quality of a bulk drug material, much more hormone-like structures in European Pharmacopoeia 4th ed. important than the assay, especially if the latter is carried out by (Ph. Eur. IV),6 United States Pharmacopoeia 26th ed. (USP non-specific methods. XXVI)7 and the 14th edition of the Japanese Pharmacopoeia As can be seen in Table 1, with two exceptions (conjugated (Ph. Jp. XIV).8 and esterified , where gas chromatography is used), As can be seen in Table 1, the most frequently used assay this test is carried out in all cases by semi-quantitative TLC or method in Ph. Eur. IV. is UV spectrophotometry at about 240 quantitative (mainly but not exclusively reversed phase) HPLC nm for steroids with strongly absorbing 4-ene-3-oxo-or 1,4- methods. The proportion of HPLC tests for related impurities in diene-3-oxo group and at about 280 for estrogens with a - bulk steroids in various pharmacopoeias is quite different: about type ring A with its weak, but characteristic, spectra. This 75% in Ph. Eur. IV, 50% in USP XXVI and 10% in Ph. Jp. method is naturally non-specific: the overwhelming majority of XIV. It is amazing that in many cases the USP XXVI and Ph. the impurities are measured together with the main component. Jp. XIV do not contain tests for related impurities (27 and 10%, The predominant method in the United States Pharmacopoeia is respectively). highly specific reversed phase (in some cases normal phase) In the case of HPLC tests, a UV detector is exclusively used at HPLC. In the overwhelming majority of cases, porous the wavelength of the maximum of the main component or at octadecylsilica support (3 – 10 µm) is used for the RP and 254 nm. For the thin-layer chromatographic purity test in the porous silica support (5 – 10 µm) for the NP separations. The majority of cases, sorbent layers are used that are impregnated wavelength of the UV detector is set most often at 254 nm, with dyes strongly fluorescing when irradiated at 254 nm by a which is not the maximum of the spectrum of 4-ene-3-oxo- or mercury lamp. Since most of the steroid hormone drugs 1,4-diene-3-oxosteroids. This is attributable to the early period strongly absorb UV light at this wavelength, the drugs and their in the history of HPLC when the majority of instruments were impurities appear as dark spots in the chromatogram suitable for equipped with only a mercury lamp. In the Japanese their semi-quantitative estimation. These appear as “TLC 254 Pharmacopoeia the HPLC and UV spectrophotometric assay nm” in Table 1. In some cases the use of various visualizing methods are almost equally represented. It is to be noted that reagents is necessary. Reagents containing sulfuric acid are

UV spectrophotometry around 240 nm with limits for the most frequently used. “TLC–H2SO4” in Table 1 means spraying specific absorbance is often part of the monographs as an with this reagent, heating and visual inspection of the plate and identification test, even in those cases when the assay is carried the use of long-wavelength UV light (366 nm). Other out by HPLC methods. visualizing reagents are phosphomolybdic acid It is interesting to note, and difficult to explain that in some (TLC–phosph.mol.), p-toluenesulfonic acid, vanillin/sulfuric instances visible spectrophotometric methods, such as a acid (TLC–vanillin), potassium dichromate/sulfuric acid measurement around 380 nm after condensation of the (TLC–acid dichromate), iodine vapor (TLC–I2) and alkaline unsaturated 3-oxo group with isoniazide, or an indirect Blue Tetrazolium (TLC–tetrazolium). With a few exceptions, measurement at 525 nm after a reaction with Tetrazolium Blue both the TLC and HPLC tests express the impurities as the main of with reducing side chain, is prescribed by the component, which can be source of serious errors (under- or European and United States Pharmacopoeias; moreover, in one overestimation) if the chromophoric system or the color and/or instance ()7 the assay is based on a non-stoichiometric the intensity of the TLC spot of the main component and the color reaction with a –sulfuric acid reagent (545 nm). impurity are different. The selectivity of these, rather outdated methods2–4,9,10 is not The use of other methods, such as the extraction of free better than that of the spectrophotometric methods based on corticosteroids from the solution of their water soluble , or native absorbance measurements. elution of TLC spots followed by UV spectrophotometric Although the overwhelming majority of steroid hormone measurement or gas chromatography, is restricted to a few drugs do not posses functional groups suitable for titration, in cases. some cases this method is still used. Ethinylsteroids can be titrated with sodium hydroxide after a reaction with silver 2·3 Assay of steroid hormone formulations nitrate and the liberation of nitric acid. This method is used as a In this section the state-of-the-art in the field of determining (non specific) assay method by Ph. Eur. and Ph. Jp. and as a the active ingredient content of steroid hormone formulations is “Limit of ethynyl group” by the United States Pharmacopoeia. based mainly on USP XXVI, since the European Other titrimetric methods are restricted to the titration of Pharmacopoeia does not contain monographs for formulations ANALYTICAL SCIENCES MAY 2004, VOL. 20 769

Table 1 Assay and related impurities tests in European, USP and Japanese pharmacopoeias

Assay Related impurity

Ph. Eur. 4 USP XXVI Ph.Jp. XIV Ph. Eur. 4 USP XXVI Ph. Jp XIV

Alclometasone — RP-HPLC 254 nm — — TLC 254 nm — dipropionate — RP-HPLC 254 nm — — — — Beclomethasone VIS–tetrazolium RP-HPLC 254 nm RP-HPLC 254 nm RP-HPLC 254 nm — TLC–tetrazolium dipropionate UV 238.5 nm RP-HPLC 240 nm RP-HPLC 240 nm TLC–H2SO4 TLC–H2SO4 TLC 254 nm - UV 240 nm RP-HPLC 254 nm — RP-HPLC 254 nm TLC 254 nm — -benzoate — RP-HPLC 254 nm — — TLC 254 nm — -dipropionate UV 240 nm RP-HPLC 254 nm UV 239 nm RP-HPLC 254 nm RP-HPLC 254 nm TLC 254 nm -sodium UV 241 nm RP-HPLC 254 nm RP-HPLC 254 nm RP-HPLC 254 nmBetamethasone– TLC 254 nm phosphate extraction 239 nm -valerate UV 240 nm RP-HPLC 254 nm RP-HPLC 254 nm RP-HPLC 254 nm RP-HPLC 254 nm TLC–tetrazolium RP-HPLC 240nm — — RP-HPLC 240 nm — — — — UV 285 nm — — RP-HPLC 236nm acetete — RP-HPLC 240 nm — — RP-HPLC 240 nm — propionate UV 235 nm — — RP-HPLC 241 nm — — butyrate — VIS–isoniazid — — TLC/elut 238 nm — pivalate acetate UV 237 nm RP-HPLC 254 nm — RP-HPLC 254 nm RP-HPLC 254 nm — UV 282 nm — — RP-HPLC 254 nm — — acetate

Danazol — UV 285 nm — — TLC–I2 — Desoxycort(icoster) UV 240 nm VIS–tetrazolium — RP-HPLC 254 nm — — one acetate -pivalate — RP-HPLC 254 nm — — — — — RP-HPLC 254 nm — — — — UV 238.5 nm RP-HPLC 254 nm RP-HPLC 254 nm RP-HPLC 254 nm RP-HPLC 254 nm TLC 254 nm -acetate UV 238.5 nm RP-HPLC 254 nm — RP-HPLC 254 nm RP-HPLC 254 nm — -sodium UV 241.5 nm RP-HPLC 254 nm — RP-HPLC 254 nm RP-HPLC 254 nm — phosphate — NP-HPLC 254 nm — — NP-HPLC 254 nm — diacetate — — GC — — TLC–vanillin propionate — RP-HPLC 280 nm — — RP-HPLC 280 nm — UV 238 nm NaOH RP-HPLC 205 nm — RP-HPLC 280 nm NP-HPLC 280 nm — -benzoate UV 231 nm — RP-HPLC 230 nm RP-HPLC 230 nm — TLC 254 nm -cypionate — RP-HPLC 280 nm — — — — -valerate UV 280 nm RP-HPLC 280 nm — RP-HPLC 220 nm TLC–H2SO4 — UV 281 nm UV 281 nm RP-HPLC 280 nm NP-HPLC 254 nm TLC–H2SO4 TLC–H2SO4 Estrogens, GC GC — GC GC — , — GC — — GC — esterified

Estrone — RP-HPLC 280 nm — — TLC–H2SO4 — — RP-HPLC 213 nm — — RP-HPLC 213 nm — Titration ethinyl RP-HPLC 280 nm Titration ethinyl RP-HPLC 280 nm — –colorim. Ethynodiol — RP-HPLC 200 nm — — RP-HPLC 200 nm — diacetate RP-HPLC 210 nm RP-HPLC 215 nm — RP-HPLC 210 nm RP-HPLC 210 nm — UV 238 nm VIS–tetrazolium — RP-HPLC 254 nm TLC 254 nm — acetate

Flumetasone UV 239 nm VIS–tetrazolium — RP-HPLC 254 nm TLC–H2SO4 — pivalate — RP-HPLC 254 nm — — TLC 254 nm — UV 238 nm RP-HPLC 254 nm RP-HPLC 254 nm RP-HPLC 238 nm — RP-HPLC 254nm acetonide — RP-HPLC 254 nm RP-HPLC 254 nm — RP-HPLC 254 nm TLC–tetrazolium UV 242 nm — — RP-HPLC 243 nm — — pivalate — RP-HPLC 254 nm RP-HPLC 254 nm — — TLC 254 nm Continued 770 ANALYTICAL SCIENCES MAY 2004, VOL. 20

Fluoxymesterone — RP-HPLC 254 nm NP-HPLC 254 nm — RP-HPLC 254 nm TLC 254 nm Flurandrenolide — RP-HPLC 240 nm — — TLC 254,366 nm — RP-HPLC 239 nm — — RP-HPLC 239 nm — — propionate — UV 239 nm — — TLC/elut. 239 nm — UV 241.5 nm NP-HPLC 254 nm NP-HPLC 254 nm RP-HPLC 254 nm NP-HPLC 254 nm TLC 254 nm -acetate UV 241.5 nm NP-HPLC 254 nm RP-HPLC 254 nm RP-HPLC 254 nm RP-HPLC 254 nm TLC–tetrazolium -butyrate — RP-HPLC 254 nm UV 241 nm — RP-HPLC 254 nm TLC–tetrazolium -hydrogen UV 241.5 nm NP-HPLC 254 nm — RP-HPLC 254 nm RP-HPLC 254 nm — (hemi)succinate -sodium — Enzime, extraction RP-HPLC 254 nm — Hydrocortisone, RP-HPLC 254nm phosphate 239 nm extraction 239 nm -sodium — VIS–tetrazolium UV 240 nm — — TLC 254 nm succinate -for — NP-HPLC 254 nm — — NP-HPLC 254 nm — -succinate — — RP-HPLC 254 nm — — TLC 254 nm -valerate — RP-HPLC 254 nm — — — — Hydroxyprogesterone — UV 240 nm — — TLC–H2SO4 — caproate — NP-HPLC 254 nm — — RP-HPLC 254 nm — acetate Titration ethinyl UV 241 nm — TLC–phos.mol. TLC–phos.mol. — Titration ethinyl — — TLC–H2SO4 — — Medroxyprogester- UV 241 nm RP-HPLC 254 nm — RP-HPLC 254 nm RP-HPLC 254 nm — one acetate acetate UV 287 nm RP-HPLC 280 nm — RP-HPLC 254 nm — — — — RP-HPLC 265 nm — — TLC–H2SO4 Meprednisone — TLC 238 nm extr. — — — — RP-HPLC 254 nm — — RP-HPLC 200 nm — — TLC–p-toluene- sulfonic acid Mestranol Titration ethinyl VIS–H2SO4 UV 279 nm TLC–H2SO4 — TLC–H2SO4 — — UV 242 nm — — TLC 254 nm acetate Metenolone — — UV 242 nm — — TLC 254 nm enanthate Methylprednisolo- UV 243 nm NP-HPLC 254 nm UV 243 nm TLC 254 nm RP-HPLC 254 nm TLC–tetrazolium ne -acetate UV 243 nm — — RP-HPLC 254 nm — — -hydrogen UV 243 nm — — RP-HPLC 254 nm — — succinate UV 241 nm RP-HPLC 241 nm UV 241 nm TLC 254 nm RP-HPLC 254 nm TLC 254 nm — RP-HPLC 254 nm — — — — UV 249 nm RP-HPLC 254 nm — RP-HPLC 254 nm TLC 254 nm — furoate Nandrolone — RP-HPLC 240 nm — — NP-HPLC 238 nm — decanoate Nandrolone — TLC 239 nm extr. — — — — phenylpropionate

Norethisterone Titration ethinyl UV 240 nm Titration ethinyl TLC–H2SO4 TLC–H2SO4 — (norethindrone) -acetate Titration ethinyl UV 240 nm Titration ethinyl RP-HPLC 254 nm RP-HPLC 254nm

Norethynodrel — UV 240 nm–HCl — — TLC–H2SO4 — — RP-HPLC 244nm — — RP-HPLC 244 nm — NP-HPLC 210 nm Titration ethinyl UV 241 nm Titration ethinyl TLC–phos.mol. TLC–phos.mol. TLC 254 nm Oxandrolone — Titration lactone — — TLC–H2SO4 — — TLC 315 nm extr. — — — — Pancuronium Titration HClO4 — Titration HClO4 TLC–I2 — TLC–NaNO2+BiI3 bromide

Prasterone sodium — — Ion-exchange/ — — TLC–H2SO4 Titration NaOH RP-HPLC 243 nm — — RP-HPLC 243 nm — — Prednisolone UV 243.5 nm NP-HPLC 254 nm RP-HPLC 247 nm RP-HPLC 254 nm TLC 254,366 nm TLC–tetrazolium -acetate UV 243 nm NP-HPLC 254 nm RP-HPLC 254 nm RP-HPLC 254 nm NP-HPLC 254 nm TLC 254 nm -hemisuccinate — TLC 243 nm extr. — — — — -pivalate UV 243 nm — — RP-HPLC 254 nm — — -sodium UV 247 nmUV 241 nm — RP-HPLC 254 nmPrednisolone — Continued ANALYTICAL SCIENCES MAY 2004, VOL. 20 771

phosphate enzime, extract. extraction 241 nm -sodium — VIS–tetrazolium RP-HPLC 254 nm — — — succinate -succinate — — RP-HPLC 242 nm — — TLC 254 nm -tebutate — NP-HPLC 254 nm — — — — UV 238 nm RP-HPLC 254 nm — RP-HPLC 254 nm NP-HPLC 254 nm — UV 241 nm RP-HPLC 254 nm UV 241 nm RP-HPLC 241 nm — TLC 254 nm — RP-HPLC 242 nm — — RP-HPLC 242 nm — UV 238 nm RP-HPLC 254 nm UV 238 nmRP-HPLC TLC–H2SO4 TLC–H2SO4 254/283 nm Titration HClO4 Titration HClO4 — TLC–H2SO4 TLC–H2SO4 — — VIS–isoniazid — — TLC 254 nm + — acid dichromate Testosterone UV 241 nm TLC 239 nm extr. — TLC 254 nm — — -cypionate — GC — — — — -enantate UV 241 nm VIS–isoniazid UV 241 nmTLC–H2SO4 TLC–p-toluene — RP-HPLC 240 nm sulfonic acid -propionate UV 241 nm VIS–isoniazid UV 241 nm TLC 254 nm — TLC 254 nm acetate — RP-HPLC 344 nm — — TLC–phos.mol. — RP-HPLC 344 nm UV 238 nm RP-HPLC 254 nm RP-HPLC 254nm RP-HPLC 238 nm — — -acetonide UV 238.5 nm RP-HPLC 254 nm RP-HPLC 254nm RP-HPLC 254 nm RP-HPLC 254 nm TLC 254 nm -diacetate — RP-HPLC 254 nm — — — — -hexacetonide UV 238 nm RP-HPLC 254 nm — RP-HPLC 254 nm RP-HPLC 254 nm —

and Pharm. Jp. XIV contains only a few (the formulations in and valerate injection, estriol tablets, estrone Pharm. Jp. XIV are indicated by cursive letters). injectable , estropipate tablets and vaginal cream, A great variety of sample-preparation methods are used to ethynodiol diacetate and mestranol tablets, ethynodiol diacetate assay the formulations. Solid dosage forms (tablets, etc.) are and ethinylestradiol tablets, finasteride tablets, fludrocortisone either disintegrated with water, followed by dilution with acetate tablets, flunisolide nasal solution, methanol, acetonitrile or the mobile phase of the RP-HPLC cream, ointment and topical solution, fluocinonide cream, system, or extraction of the aqueous phase with . ointment, gel and topical solution, fluorometholone ophthalmic More common is direct extraction mainly with methanol. suspension, tablets, flurandrenolide cream, Direct extraction with methanol, chloroform, acetonitrile, etc. is lotion, tape, halcinonide cream and ointment, hydrocortisone the most widely used sample-preparation method for creams, cream, ointment, gel, lotion, rectal suspension, hydrocortisone while in addition to these, extraction with hexane, heptane, i- butyrate cream, cream and ointment, octane followed by extraction of the apolar phase with mixtures isofluprednone acetate injectable suspension, levonorgestrel and of water and methanol, acetonitrile, etc. is also used for ethinylestradiol tablets, acetate tablets, ointments. Aqueous solutions are usually diluted with water or oral suspension and tablets, the mobile phase of the RP-HPLC method, or extracted with tablets, methylprednisolone acetate chloroform, ethyl acetate etc. Aqueous suspensions are injectable suspension, mometasone furoate cream, ointment and homogenized by simple dilution with methanol, 2-propanol or topical solution, injection, norethindrone the mobile phase of the RP-HPLC system. Oily injections are and ethinylestradiol tablets, norethindrone and mestranol either simply diluted, mainly with chloroform or diluted with tablets, norgestrel and ethinylestradiol tablets, prednisolone oral hexane, etc., followed by extraction with polar solvents or suspension and syrup, prednisolone tablets, prednisolone solvent mixtures. acetate injectable suspension and ophthalmic suspension, The predominant method for the assay is reversed phase prednisone oral solution, injectable suspension and tablets, HPLC with UV detection, either at 254 nm or at the maximum progesterone injection, injectable suspension and vaginal of the UV spectra of the active ingredients. This method is used suppositories, rimexolone ophthalmic suspension, spironolactone among others for the assay of aclometasone dipropionate and tablets, spironolactone and hydrochlorothiazide tablets, amcinonide cream and ointment, betamethasone cream and triamcinolone tablets, cream, ointment, tablets, gel, betamethasone topical aerosol, lotion and injectable suspension, triamcinolone dipropionate cream, ointment, lotion and topical aerosol, diacetate oral suspension, syrup and injectable suspension and betamethasone sodium phosphate injection, betamethasone triamcinolone hexacetonide injectable suspension. sodium phosphate and acetate injectable suspension, Normal-phase HPLC is used for the assay of cream, ointment and lotion, clobetasol tablets, diflorasone cream, ointment, propionate cream, ointment and topical solution, cortisone fluorometholone cream, hydrocortisone tablets, injectable suspension, capsules, acetate cream, ointment and lotion, medroxyprogesterone desoxycorticosterone pivalate injectable suspension, acetate injectable suspension, prednisolone tablets and desoxymetasone cream and ointment, dexamethasone injection, injectable suspension. ophthalmic suspension, oral solution elixir and tablets, Of the other chromatographic methods, gas chromatography is injectable suspension, dexamethasone used for the assay of drostanolone propionate injection, sodium phosphate cream, injection, ophthalmic ointment and esterified and conjugated tablets, mibolerone oral ophthalmic solution, dydrogesterone tablets, estradiol tablets solution, oxandrolone tablets and and vaginal cream, injectable suspension, injection. TLC separation, extraction followed by Tetrazolium 772 ANALYTICAL SCIENCES MAY 2004, VOL. 20

Blue colorimetry is the assay method for betamethasone syrup determination of the active ingredients of two- or and methylprednisolone acetate cream, while column multicomponent formulations. In simple cases improved chromatographic separation followed by Tetrazolium Blue extraction procedure such as solid phase extraction in the case colorimetry or iron-phenol colorimetry is used for the assay of of the determination of hydrocortisone in ointments12 is dexamethasone gel and estradiol pellets and injectable sufficient. Dual-wavelength measurements can also give suspension, respectively. satisfactory results. For example, the release of hydrocortisone The above-discussed chromatographic methods are selective acetate from microcapsules in aqueous suspension was and stability indicating. The same cannot be said for the non- monitored at 247.6 nm with 277 nm as the reference chromatographic methods. UV spectrophotometric assay based wavelength.13 The classical dual-wavelength spectrophotometry on natural absorption is carried out for dexamethasone sodium was successfully used for the simultaneous determination of phosphate inhalation aerosol, dydrogesterone tablets, spironolactone and hydrochlorothiazide with their overlapping methyltestosterone tablets and capsules, oxymetholone tablets, spectra in a formulation.14 The performance of the prednisolone sodium phosphate injection and ophthalmic method can be improved by multiwavelength measurements and solution (after enzymatic hydrolysis and extraction), chemometric methods. Partial least-squares regression analysis progesterone in intrauterine contraceptive system, stanozolol was applied to other diuretic preparations, such as tablets and testosterone injectable suspension. A UV spironolactone–althiazide15 and spironolactone–chlorthalidone16 spectrophotometry is used in some other cases as well in the formulations. The much more delicate problem of the assay of content uniformity and dissolution tests of tablet formulations. the low-dosed levonorgestrel–ethinylestradiol17 and The selectivity of some colorimetric methods also used for the –ethinylestradiol18 oral contraceptive tablets was also assay of steroid hormone formulations is not better either.2–4,9,10 solved by the partial least-squares regression analysis. Even For example, reactions based on the condensation of the three-component drug formulations were successfully analyzed unsaturated 3-oxo group with isoniazid ( by this and other chemometric methods, such as the principal cream, hydrocortisone caproate injection, nandrolone component regression method. One example is the mixture of phenylpropionate injection, norethindrone tablets, norgestrel hydrocortisone, and oxytetracycline.19 The accuracy tablets, testolactone tablets, injection and and precision of the determination of even as low a quantity as 2 injection) or 4-aminoantipyrine mg/vial dexamethasone in the presence of 250 and 10 mg of

(flumethasone pivalate cream) measure the less vulnerable part vitamins B6 and B12, respectively, in an injection formulation of the . The reactions of the phenol-type ring A based were good.20 Other 3-component formulations containing low on diazo-coupling (estradiol benzoate injection) or on the non- quantities of dexamethasone resolved by partial least-squares stoichiometric reaction with the sulfuric acid–methanol reagent regression analysis and other chemometric methods were (ethinylestradiol tablets) are not stability-indicating either. The , trimethoprim, dexamethasone21 and situation is somewhat better with the color reactions of chlorpheniramine, and dexamethasone.22 In the corticosteroids with sensitive, reducing side chains at position latter case, the use of artificial neural network analysis was 17. Most of their degradation products do not react with the necessary for the determination of dexamethasone due to the alkaline Tetrazolium Blue (desoxycorticosterone acetate non-linearity caused by interactions among the components. injection and pellets, dexamethasone topical aerosol, Derivative UV spectrophotometry10 was also successfully hydrocortisone injectable suspension and hydrocortisone acetate applied to the analysis of steroid hormone formulations. In ophthalmic ointment and suspension, injectable suspension) or simple cases (single-component formulations) only the spectral phenylhydrazine–sulfuric acid reagents (hydrocortisone sodium background caused by the should be eliminated. For phosphate injection and prednisolone cream). this purpose first-derivative spectra are usually sufficient. An It is amazing that an extremely outdated and labor-extensive example for this is the determination of triamcinolone acetonide method is used by USP XXVI for the assay of estrone oily in an ointment.23 The same technique using the “zero-crossing” injection. The procedure includes three extraction steps method was found to be suitable for the simultaneous (consuming 50 ml of hexane, 50 ml of benzene (!) and 95 ml of determination of the components of dual-component drug chloroform), two evaporations to dryness, formation of formulations such as creams and tablets containing hydrazone derivative with trimethylacethydrazide triamcinolone acetonide and terbinafine hydrochloride24 and chloride and its decomposition. Finally the weight of estrone is oral contraceptives containing ethinylestradiol and measured! levonorgestrel25,26 as well as ethinylestradiol and gestodene.27 A zero-crossing second-derivative method was used for the determination of fluorometholone and tetrahydrozoline 3 Spectroscopic and Spectrophotometric Methods hydrochloride28 as well as estradiol and medroxyprogesterone acetate29 in formulations. Another possibility to improve the 3·1 Ultraviolet-visible spectrophotometry selectivity of the method is the use of “ratio spectrum (zero- As shown in the previous sections, in spite of its poor cross) derivative spectroscopic” method. Examples for this are selectivity UV spectrophotometry is widely used for the the determination of spironolactone and hydrochlorothiazide,14 identification and assay of bulk steroid drugs. The assay is hydrocortisone, nystatin and oxytetracycline,19 cyproterone carried out either using a reference standard material or by acetate and estradiol valerate30 in pharmaceutical formulations. determining and describing the value of the specific absorbance. The performance of various multivariate chemometric and The validation of the determination of the latter by derivative spectrophotometric methods is compared for interlaboratory study preceded by recrystallization and checking examples of formulations containing hydrocortisone and Zn- the purity of the test sample by phase solubility analysis, HPLC Bacitracin31 as well as dexamethasone and polymyxin B.32 An and differential scanning calorimetry was described on the interesting feature of another method for the assay of example of triamcinolone.11 Problems to be solved for the spironolactone and hydrochlorothiazide, based on ratio application of this method to the assay of drug formulations is spectrum derivative spectroscopy, making use of a rapid- the elimination of matrix effects and simultaneous scanning diode-array spectrophotometer, is that it is adopted to ANALYTICAL SCIENCES MAY 2004, VOL. 20 773 flow-injection analysis.33 of identity, as well as the content of various drugs, among The importance of spectrophotometric/colorimetric methods others finasteride in their solid formulations.45 TG/FT-IR based on chemical reactions has dramatically decreased in the studies supported by X-ray diffraction and DSC revealed the last years both in the analysis of bulk steroid drugs and mechanism of the decomposition of prednisolone hemisuccinate formulations made thereof. As can be seen in Table 1 and in the in a freeze-dried formulation.46 section “Assay of steroid hormone formulations” some of the Near-infrared reflectance spectroscopy has become a versatile classical reactions (e.g. condensation with isoniazid and tool in drug analysis and in-process control in drug production. phenylhydrazine, reaction with tetrazolium reagents) are It was found to be suitable to solve as delicate a problem as the still in use in some instances. Many other reactions were also simultaneous determination of and described.2–4,9,10 Further research in this field can be considered ethinylestradiol in oral contraceptive tablets.47 to be unnecessary: publications of this kind published in the last 15 years are not referred to in this review unless these are 3·4 Mass spectrometry particularly interesting. This is the case with a paper where In the majority of cases, mass spectrometry is applied in Friedel–Crafts of steroids followed by taking the UV pharmaceutical steroid analysis associated with various spectrum enables interesting discrimination to be made between chromatographic techniques. Examples for these will be various isomeric derivatives.34 Kinetic variants of the classical presented in some of the subsequent sections. Here, only one Blue Tetrazolium and phenylhydrazine35 and isoniazid36 direct application is mentioned. Time-of-flight secondary ion reactions and their application to the analysis of corticosteroids mass spectrometry was successfully applied for the in various pharmaceuticals are also worth mentioning. characterization and imaging of a controlled-release drug delivery system containing prednisolone sodium 3·2 Fluorimetry metasulfobenzoate. Cross sections of the device containing a The native fluorescence of estrogens enables their direct multi-polymer-layer system were probed for the distribution of fluorimetric determination, e.g. the determination without a the active ingredient.48 preliminary separation of in an injection formulation in the presence of hydroxyprogesterone caproate 3·5 NMR spectroscopy (Ex: 295 nm/Em: 308 nm).37 However, the main application One of the relatively rare direct applications of NMR field of this is their sensitive HPLC detection. spectroscopy in pharmaceutical steroid analysis is its use for the Measurements based on fluorescence induced by non- rapid screening and compositional analysis of steroid cocktails stoichiometric reactions with various strongly acidic reagents3,4,9 and veterinary drug formulations illegally used to livestock. have almost completely lost their importance in modern Stanozolol, fluoxymesterone, methylboldenone, pharmaceutical analysis. However, as seen in the sections , clobestol acetate, testosterone and its dealing with pharmacopoeias, methanol–sulfuric acid is still an cypionate and decanoate and other steroids were identified important spray reagent in the thin-layer chromatographic directly or after HPLC fractionation with the aid of a 1H-NMR analysis of steroid drugs. An interesting, highly specific and database.49 When using interrupted decoupling, solid state 13C- sensitive FIA method for the determination of fluticasone NMR is suitable not only for detecting low-dose active propionate is based on splitting with sodium hydroxide of its ingredients (among others prednisolone) in solid dosage forms,

–CO–S–CH2F side chain to form fluoromethylthiol which is but also for discrimination between the two polymorphic forms further reacted with glycine and o-phthalaldehyde. The strongly of the latter.50 Further analytical applications of NMR fluorescent isoindole derivative is formed in the reaction coil of spectroscopy are discussed in the section dealing with HPLC. the FIA apparatus.38 In addition to analytical applications, NMR spectroscopy is a good tool for studying the mechanism of interactions taking 3·3 Infrared and Raman spectroscopy place between the analyte and stationary phase. As an example, Infrared (IR) spectroscopy is the most generally used method the transferred NOESY study of the interactions between for the identification of bulk steroid drugs in modern various steroids and calix[8]arene-based stationary phases is pharmacopoeias.6–8 In addition to this, IR spectroscopy, mentioned.51 Similar studies related to chiral interactions are especially Fourier-transform IR (FTIR) is one of the most shown in the last section of this review. important methods, together with X-ray powder diffractometry, differential scanning calorimetry (DSC), thermogravimetry 3·6 Chemiluminometry (TGA) and thermomicroscopy to characterize the solvates and Although the main application field of chemiluminescence in polymorphic modifications frequently occurring among steroid steroid analysis is chemiluminescence immunoassay in hormone drugs.39 One of the most characteristic examples is biological-clinical analysis, there are also a few direct spironolactone, where in the early literature several polymorphs applications in pharmaceutical analysis. The sensitising effect and solvates were described. Conventional IR spectrometry of corticosteroids on the chemoluminogenic reaction between using fused KBr discs is not suitable for their differentiation due sulfite and cerium(IV) was exploited for their flow-injection to the easy transformation of the polymorphs under pressure. (FIA) determination in pharmaceutical formulations.52,53 A On the basis of diffuse reflectance infrared Fourier transform luminol chemiluminescence determination for the same purpose spectroscopy (DRIFTS) and Fourier transform Raman is also described.54 spectroscopic investigations supported by DSC and TGA measurements40 four different polymorphic forms41 and five 3·7 Circular dichroism (CD) spectropolarimetry solvates42 were identified and characterized. FT-IR studies The main application field of CD spectroscopy is structural combined with all of the above mentioned solid-phase analysis but this technique can be successfully used also for techniques revealed the existence of three polymorphs of analytical purposes. The early literature of the applications to fluocinolone acetonide43 as well as two polymorphs and a steroid hormone analysis was reviewed.55 Of the recent hemihydrate of flunisolide.44 applications, two are mentioned here. The profound difference The DRIFTS method is also suitable for the rapid verification between the ellipticities of and its 5-ene isomer 774 ANALYTICAL SCIENCES MAY 2004, VOL. 20 enables their selective determination (ethisterone at 348 nm at hydrocortisone, hydrocortisone acetate and its negative maximum of the CD spectrum) and the isomer at containing ointment and cream preparations was fluorescence 296 nm at the positive maximum of the CD curve. When the quenching at 254 nm.73 In the case of estradiol tablets (284 simultaneous determination was carried out by HPLC, the nm), the HPTLC method was suitable not only for the assay but spectropolarimeter could act as a CD-detector.56 The also for the detection of degradants formed under stress applicability of the CD method can be enhanced by the use of conditions.74 preliminary chemical reactions. A difference CD method was Reversed-phase (ion pair) TLC74 and HPTLC75 systems were developed for the determination of 4-ene-3-oxo steroids based used for the assay of sodium phosphate salts in on formation making use of the large difference between parentheral preparations and ear drops75 and various steroids,76 the ellipticities before and after oximation. This method was respectively. found to be suitable for the determination to as low a Overpressured thin-layer chromatography (OPLC) is a variant concentration as 0.02% of impurity in of HPTLC in which the sorbent layer is covered with a norgestimate.57 pressurized elastic plate thus eliminating the vapor phase. Due to the forced flow of the eluent delivered by an automatic pump its migration velocity is constant and the migration distance can 4 Chromatographic and Hyphenated Methods be increased up to 18 cm thus enabling excellent separations and improved quantifiability to be achieved. As a result of 4·1 Planar chromatography these factors, OPLC is eminently suitable for the separation of In contrast to the pharmacopoeias where TLC is used only as impurities and degradants in bulk drugs and their formulations. a semi-quantitative limit test for purity check of bulk steroid Examples for the application of OPLC to steroid hormone drugs hormone drugs, in the literature of the last 15 years almost are the purity test of norgestrel and levonorgestrel (visualization exclusively quantitative densitometric methods are described for by fluorescence quenching at 254 nm, phosphomolybdic acid or the purity check and also for the assay of steroid hormone sulfuric acid),77 drug substance and tablet (sulfuric formulations. Typical papers describing the assay of acid),78 nandrolone (sulfuric acid; comparison of the impurity formulations are e.g., the determination of betamethasone profile with those obtained by TLC, HPLC and GC),79 valerate in various creams together with other active norethisterone drug substance and tablet (sulfuric acid).80 ingredients. Conventional silica gel 60 F254 plates were used; OPLC was successfully used in in-process-monitoring81 and (see section “Related impurities test of bulk drug materials”). cleaning validation of equipment82 in the course of the The densitograms were scanned in the reflectance mode at production of bulk steroids and steroid formulations. wavelengths which correspond to the isobestic point of the two The usefulness of further innovations, such as temperature- components: 228 nm for betamethasone– cream,58 controlled programmed TLC,83 programmed multiple 247 nm for betamethasone–clioquinol cream59 and 233 nm for development TLC with a new modification of the horizontal betamethasone– nitrate cream.60 The determination sandwich chamber84 and ultra-thin-layer chromatography of the six components of conjugated estrogens in raw materials (UTLC) where the granular adsorbents are replaced by a and tablet formulations was carried out after hydrolysis, monolithic structure based on a silica-gel matrix has been extraction, followed by TLC separation and detection at 280 demonstrated by using, among others, the separation of steroid nm.61 mixtures.85 An interesting study dealing with the reasons for If carefully validated, the quantitative TLC densitometric and the elimination of the irreversible adsorption of some purity test is equivalent to the HPLC purity test. The principles steroids during multiple development TLC merits mentioning.86 and practice of validation were described taking danazol62 and A tandem TLC-HPLC method is described for screening estradiol benzoate63 as examples (scanning at 252 and 229 nm, cosmetic products for the presence of undeclared synthetic respectively). corticosteroids.87 Chiral selectors improved the separation of The popularity of high-performance thin-layer diastereomeric steroid pairs, e.g., the addition of β-cyclodextrin chromatography (HPTLC) is continuously increasing: in the to the mobile phase in the case of the separation of R(+)- and majority of papers published in the last 15 years the use of this R(–)-budenoside on cellulose-coated HPTLC plates,88 and method is reported. The advantages of this technique are clear: impregnation of the silica gel TLC plate with d- due mainly to the smaller particle size (about 4 µm) and narrow camphorsulfonic acid/copper(II) acetate for the separation of the particle-size distribution of the stationary phases, the separation diastereomeric 21- derivatives of prednisolone formed with efficiency increases, enabling well-separated and well- racemic tetrahydrophthalic acid.89 quantifiable spots to be achieved with smaller plates, shorter Some papers deal with the possibilities of applying only running distance and shorter running time. Some of the seldom-used stationary phases e.g., Florisil, derivatized important parameters (retention behavior, separation efficiency, reversed-phase systems for steroid separations and with the selectivity) of TLC and HPTLC were compared using steroids elucidation of the separation mechanism.90,91 as the model compounds.64 In the majority of cases, silica gel 60 F254 plates were used for the assay of steroid preparations. 4·2 Gas chromatography (GC) and GC-MS Some of the investigated formulations are as follows (the As already mentioned earlier in this review, and as seen in wavelength of the densitometric scan in parentheses): Table 1, gas chromatography does not play a very important finasteride tablets (228 nm),65 betamethasone tablets (245 nm),66 role in the analysis of steroid hormone drugs. The reasons for mometasone furoate topical preparations (260 nm),67 this are their low volatility and (especially in the case of –ethinylestradiol tablets (284 nm)68 or corticosteroids) thermal instability. In the case of sufficiently ethinylestradiol (220 nm) and cyproterone acetate (284 nm),69 stable steroids it is a good method of choice for the assay of dexamethasone and in nasal drops (240 nm),70 among others methyltestosterone (after dimethylethyl- or dexamethasone sodium phosphate– sulfate solution dimethylisopropylsilylation)92 and cyproterone acetate– (246 nm),71 in pharmaceutical formulations ethinylestradiol93 tablets. (243 nm; stepwise gradient).72 The basis for the assay of GC-MS is an ideal tool for screening purposes. As many as ANALYTICAL SCIENCES MAY 2004, VOL. 20 775

134 underivatized drugs (among them 10 steroids) were micellar systems and other formulations,128 triamcinolone, its included in a study to screen ethnic patent available acetate and acetonide and several other corticosteroids (St),129 in health-food stores and ethnic markets. It is to be noted that triamcinolone acetonide dermatological patches,130 two of the ten (dexamethasone and prednisolone) could not be triamcinolone acetonide, methyl- and propylparaben topical detected by GC-chemical ionization MS, probably due to their cream (St).131 vulnerable 17-side chain.94 One possibility to enhance the Carefully validated stability-indicating HPLC methods were applicability of GC and GC-MS to some of the thermally labile used for the determination of impurities and degradants in bulk compounds is “supersonic GC-MS” using shorter capillaries pipecuronium bromide,132 budesonide,133 aqueous with lower film thickness, an increased carrier gas flow rate and spironolactone-β-cyclodextrin solutions,134 and for the a decreased temperature. This enables several steroids (among photodegradation kinetic study of danazol.135 them with a vulnerable side chain) to be HPLC is eminently suitable for screening purposes, e.g. RP- determined.95 Another screening study using GC-MS aimed at HPLC for anabolic steroids,136 corticosteroids in topical detecting anabolic steroids96 and prohormones97 in nutritional pharmaceuticals using diode-array UV detector,137 toxicological supplements. analysis of steroids and other compounds with diode-array UV detector,138 NP- and RP-HPLC for corticosteroids.139 Especially 4·3 Supercritical fluid chromatography (SFC) useful is the coupling of HPLC with mass spectrometry: a Although the problem of thermal instability in GC can be HPLC/time-of-flight MS method was developed for screening solved by using supercritical fluid chromatography, this anabolic steroids in illegal cocktails,140 while for their technique seems to have attracted little interest in steroid determination in oily formulations RP-HPLC separation analysis. Several steroids were separated by SFC with light- followed by off-line GC-MS analysis was used.141 HPLC-MS is scattering detection.98,99 The sensitivity of the laser light the most suitable method for the rapid screening of high- scattering detector is similar to that of the UV detector with the throughput drug mixtures.142 Capillary HPLC-MALDI-TOF- advantage that UV-inactive steroids can also be detected with MS after post-column derivatization with 2,4- the same sensitivity.99 Examples for the use of chiral SFC will dinitrophenylhydrazine was used for the screening of be shown in the section “Enantiomeric separations”. combinatorial libraries with 7 corticosteroids as model Supercritical fluid extraction can be a useful alternative to compounds.143 HPLC-MS was also used for the rapid classical extractions as demonstrated on the examples of identification of the products of biotechnological megestrol acetate,100 medroxyprogesterone acetate101 and transformations. The model for this study was the cyproterone acetate101 from tablet matrices. of progesterone to 9α-hydroxyprogesterone.144 HPLC-UV was used as the in-process-control method to follow 4·4 High-performance liquid chromatography (HPLC) and this reaction at the industrial scale.145 HPLC-MS On-line HPLC-NMR—although only few data are available HPLC as the most important and most generally used method for its use in pharmaceutical steroid analysis—is certainly the in pharmaceutical steroid analysis is described and method of the future even in this field. For example, using this characterized in the chapter “Steroid hormone drugs in technique estradiol and were detected in a pharmacopoeias”, where the assay and purity test for several galenic .146 Further examples for the use of HPLC bulk drugs is presented in Table 1 followed by listing several coupled with spectroscopic methods are presented in the section formulations assayed by HPLC. Some further examples are “Impurity and degradation profiling of steroid drugs”. briefly summarized here, taken from the literature where many In addition to the above discussed use of HPLC for the “pharmacopoeial-like” reversed-phase HPLC assays for analytical investigation of bulk steroids and steroid formulations formulations are described (the abbreviation “St” in parentheses it is a useful tool also in the hands of pharmaceutical relate to stability assays). Allylestrenol–α-tocopherol tablets,102 technologists to investigate drug release from various beclomethasone dipropionate inhalers103 and other formulations formulations and devices for controlled drug delivery. (St),104 betamethasone, its acetate and other corticosteroids in Examples are the release of prednisolone from chitosan gel ointments,105,106 betamethasone and dexamethasone (delicate beads147 or from chitosan-gelatin sponges,148 estradiol and its 3- separation problem!),107,108 betamethasone–diclofenac acetate from intravaginal rings149 and triamcinolone acetonide sodium–cyanocobalamine tablet,109 cyproterone acetate tablet from topical dosage forms.150 (St),110 dexamethasone–xylometazoline nasal drops,111 As mentioned earlier, in the majority of cases reversed-phase dexamethasone– palmitate ointment,112 HPLC is used, mainly C18 columns with unbuffered binary or dexamethasone–trimethoprim liquid formulations,113 ternary mixtures of water with methanol, acetonitrile or dexamethasone acetate–prednisolone tablet,114 estradiol– tetrahydrufuran. Some innovations regarding stationary phases levonorgestrel drug delivery formulation (St),115 are as follows. Monolithic columns were found useful tools for estradiol valerate–medroxyprogesterone acetate tablet,116 the ultrafast separations of steroids,151 among others ingredients ethinylestradiol–norgestrel tablet,114 ethinylestradiol– of a topical cream containing triamcinolone acetonide and levonorgestrel and ethinylestradiol–gestodene tablets,117 preservatives.152 Ultrafast analysis is also achievable by finasteride tablet (St),118 fluocortolone pivalate and hexanoate decreasing the column length with simultaneous optimization of suppositories,119 hydrocortisone–oxytetracycline–nystatin in the flow rate and gradient profile (ballistic gradients).153 pharmaceutical preparations,120 hydrocortisone acetate and Temperature-responsive stationary phases showing hydrophilic hemisuccinate– in pharmaceutical preparations,121 properties at lower while hydrophobic properties at higher hydrocortisone acetate–methyl- and propylparaben topical temperatures were prepared by covalently binding poly(N- cream (St),122 hydrocortisone sodium phosphate gel (St),123 isopropylacrylamide) to aminopropylsilica. This is a promising nestorone implants (St),124 pancuronium bromide injection tool for the temperature-controlled separation of steroids.154–156 (St),125 prednisolone acetate–tetrahydrozoline hydrochloride– Excellent separation of the epimers of estradiol was achieved by ophthalmic preparations,126 prednisolone sodium modifying the surface of silica gel with the covalent binding of phosphate implantable infusion pump (St),127 progesterone in cholesterol.157 Molecularly imprinted artificial receptors as 776 ANALYTICAL SCIENCES MAY 2004, VOL. 20

HPLC stationary phases were prepared among others for the or diode-array UV detection for various drugs including screening of corticosteroids158 and estrogens.159 Porous corticosteroids in creams187 can be considered to be curiosities. graphitic stationary phases were also used for the separation of Evaporative light scattering detector can be of importance in the corticosteroids especially if a voltage is applied to the column, case of UV-inactive steroids such as pancuronium bromide and while making use of its electric conductivity.160 This technique, related compounds.188 The native fluorescence of termed “electrochemically modulated liquid chromatography”, ethinylestradiol (ex: 285 nm/em: 310 nm) is used in the was successfully coupled on-line with electrospray MS.161 fluorimetric detection of the USP dissolution test of Several papers have been published dealing with the mobile levonorgestrel–ethinylestradiol tablets7 while terbium(III)- phase selection and optimation in the HPLC analysis of steroids sensitized fluorescence (ex: 245 nm/em: 545 nm) detection was hormone drugs. The selectivity of the separation in RP-HPLC described for various steroids in the course of their SDS was improved by using ternary systems containing methyl or micellar HPLC determination.170 ethyl acetate as demonstrated by the separation of 9α- In addition to its analytical application, HPLC is a useful tool fluoroprednisolone acetate from its impurities.162 Using 2,2,2- in other fields of steroid drug research, too. For example HPLC trifluoroethanol as a mobile phase component in ternary systems was used for the determination of apparent association constants together with perfluorinated RP-HPLC stationary phases unique of steroid-cyclodextrin inclusion complexes,189 lipophilicity selectivity was attained explicable by the strong adsorption of profiling,190 for studying drug-membrane interactions191 and the co-solvent on the surface of the stationary phase.163 Another high-throughput log P measurements.192 Biopartitioning fluorinated solvent, ethoxynonafluorobutane was introduced as micellar chromatography with polyoxyethylene (23) lauryl ether an environmentally friendly and highly selective solvent in the (Brij35) as the micelle forming agent was used for the NP-HPLC analysis of several compounds, among others prediction of human drug absorption.193 Calculations based on steroids.164 Cyclodextrins are known to be excellent chiral linear solvation energy relationship were found to be useful to selectors in enantiomeric analysis (see section “Enantiomeric find “open windows” for internal standards in RP-HPLC analysis”). The effect of β-cyclodextrin on the retention of chromatograms of various compounds, among others steroids194 steroids and their separation in general165 and the effect of and these calculations were used to find suitable internal temperature on these characteristics166 were thoroughly studied. standard for the determination of estradiol and levonorgestrel in Chromatographic data–topological index dependence of steroids transdermal drug delivery formulations.195 in RP-HPLC and RP-TLC are also worth mentioning.167 The use of micellar liquid chromatography, a special branch of RP-HPLC where the eluent contains very low concentration 5 Electrophoretic and Related Methods of organic modifiers (mainly 1-pentanol and acetonitrile) and about 0.1 M sodium dodecyl sulfate (SDS) as the micelle 5·1 Capillary electrophoresis (CE) forming agent was applied to steroid analysis by Spanish Being neutral compounds the overwhelming majority of groups.168–176 This technique enables the relatively rapid steroid hormone drugs cannot be analyzed by direct CE. This analysis of derivatives with wide range of polarity with technique is restricted to ionizable derivatives. For example, minimum need for sample preparation. In addition to retention- the simultaneous determination of hydrocortisone 21- structure relationship studies169 and screening of steroids,170–172 hemisuccinate, oxytetracycline and nystatin in pharmaceutical the method was successfully used for the determination of preparations was carried out at pH 11.196 Covalent spironolactone,168 corticosteroids,173,176 methyltestosterone,174 derivatization with electrically charged reagents, e.g. anabolic steroids176 and danazol175 in various formulations. ketosteroids with Girard P or T reagents is a solution for this Although the principles and practice of mobile phase problem: the Girard hydrazones of 4-ene-3-oxo- and 17- optimization in HPLC were already set up in the early period of oxosteroids can easily be separated and quantificated.197 the history of HPLC, further studies are time to time made Capillary electrophoresis can be a useful tool for the refining of the optimization with steroids and other compounds preconcentration of different classes of steroids, making use of as the models. These include the description of a general electroosmotic flow and capillaries partially filled with various quantitative relationship for column selectivity in RP-HPLC and reagents, e.g., borate for the complexation of steroids with the study of the effect of the change in conditions,177 the use of proximal hydroxyls, high pH buffers for weakly acidic experimental design for spherical coordinate representations of estrogens, micelle forming agents for hydrophobic steroids.198 solvent composition in RP-HPLC,178 prediction of the RP-HPLC retention of steroids using solvatochromic parameters,179 the use 5·2 Micellar electrokinetic chromatography (MEKC) of 23 factorial design and computer simulation,180 triangle MEKC is an ideal tool for the separation and quantification of optimization for the separation of finasteride and related lipophilic steroid drugs which cannot be directly analyzed by compounds,181 the effect of the pH of the mobile phase for the CE: these (mainly corticosteroids) were among the first models separation of ionisable steroids,182 the effect of column investigated after the invention of this new technique at the mid overloading on the separation of mometasone furoate and 1980s.199–201 Since then, great many papers have been published clotrimazole at widely different concentrations.183 on this topic. Based on these, it is predictable that due to the The first example for the improvements in the selectivity of minimum requirements of sample pretreatment, low costs and the UV detection in the HPLC analysis of steroids is the high speed MEKC can be a real alternative to HPLC in steroid detection of co-eluting species in e.g., danazol by subtracting analysis as demonstrated in a comparative study using the up-slope and down-slope diode-array spectra from the apex betamethasone dipropionate, clotrimazole and their related spectrum.184 HPLC-MS is an excellent tool for estimating substances as the model compounds.202 minor components under overlapping peaks, e.g., the Some of the several new applications of MEKC to identification and quantitation of as low as 0.01% prednisolone pharmaceutical steroid analysis are listed here: the in hydrocortisone.185 On-line post-column photochemical determination of ethinylestradiol and levonorgestrel203 or derivatization followed by electrochemical detection for the gestodene204 in oral contraceptives, hydrocortisone and its determination of spironolactone–hydrochlorothiazide tablets186 acetate together with various ,205 prednisolone, Zn- ANALYTICAL SCIENCES MAY 2004, VOL. 20 777 bacitracin and in ointments and ocular drops,206 MS have been reviewed with several examples from steroid prednisolone acetate, and phenylephrine in local drugs.227 The identification of impurities in fluticasone pharmaceutical preparations,207 dexamethasone, trimethoprim propionate228 and the combination of CEC with nanospray MS and polymyxin B in various formulations,208 betamethasone in with detection limits of 50 fg for corticosteroids merit special dissolution tests.209 attention.229 In the above-listed studies (and the majority of those not mentioned here) sodium dodecyl sulfate (SDS) was used as micelle forming agent in the background electrolyte. In a few 6 Electroanalytical Methods cases, bile salts were also used.199,209 A comparison of the two systems using corticosteroids as the model compounds is also A review of electroanalytical methods in steroid analysis was described.210 High concentrations of organic solvents (up to published in 1989.230 These methods have never played very 50% acetonitrile or methanol) in the background electrolyte important role in this field but sufficiently sensitive and containing SDS enables ionic () and non- selective methods are often published up to the present time ionic (beclomethasone dipropionate, 2-phenylethanol) mainly for the assay of formulations. In the majority of cases compounds to be determined within one electrokinetic run.211 differential pulse polarography, based on the reduction of the 4- Using a related method, termed hydrophobic interaction ene-3-oxo group is used, for e.g. spironolactone,231 electrokinetic chromatography seven active ingredients of an beclomethasone dipropionate,232 dexamethasone sodium ointment (among them hydrocortisone) were separated and phosphate233 and betamethasone valerate.234 Gestodene was quantitated. The migration behavior of the hydrophobic and determined in oral contraceptives by square-wave voltammetry ionic constituents was influenced by the concentration of and adsorptive stripping techniques.235 tetradecyl ammonium bromide and ammonium chloride, A direct potentiometric method was developed for muscle respectively.212 On-line sample preconcentration (370-fold relaxant quaternary ammonium compounds, among them improvement in detector response) was achieved for steroids by pancuronium bromide, based on ion-selective membrane sweeping with anionic-zwitterionic micelles.213 electrodes prepared from ion pairs with tetraphenylborate or Chiral aspects of MEKC are shown in the section dipicrylaminate as the counter in a PVC matrix.236 “Enantiomeric analysis”.

5·3 Microemulsion electrokinetic chromatography 7 Selected Analytical Tasks (MEEKC) Although in an early study better separation characteristics 7·1 Impurity and degradation profiling of steroid drugs237,238 were attained for steroids with MEEKC than with the related Impurity and degradation profiling i.e. detection, technique MEKC, this method does not seem to have been used identification/structure elucidation and quantitative as extensively in the practice as MEKC. In this study SDS or determination of impurities and degradants usually require the glycodeoxycholate were used as the micelle forming agent in complex application of one or two separation method and at MEKC and various higher alcohols, n-hexane, cyclohexane, etc. least 2 – 3 spectroscopic techniques (usually UV, MS, NMR) with SDS as the emulsion-forming solvents.214 In some further either off-line but preferably on-line. studies aiming at comparing the selectivities of the two Of the innumerable studies, a few resulting in interesting techniques among many other compounds some steroids were structures are listed here beginning with corticosteroids. Six also included.215–217 A good correlation was found between the synthesis-related impurities were identified in prednisolone.239 retention characteristics of betamethasone and its esters and the Several synthesis-related impurities240,241 and degradants242,243 218 respective log Pow values; MEEKC is a good tool for the rapid (mainly products of intramolecular oxido-reduction reactions estimation of log Pow values of a wide range of compounds, were identified in the course of the stress stability test of among them steroid hormone drugs.219 (21-deoxy-21-N-methylpiperazinyl derivative of prednisolone). Among minor impurities the 9α-bromo analogue 5·4 Capillary electrochromatography (CEC) and CEC-MS of the parent drug was found as the main impurity in CEC, which can be considered to be the combination of RP- triamcinolone acetonide.244 Of the other corticosteroids several HPLC and CE, has been successfully applied to separation and synthesis-related impurities and degradants were identified in quantitation of steroid drugs. The performance of HPLC, dexamethasone.245,246 Of these, the 17β-carboxy-17α-formyloxy MEKC and CEC was compared using six steroids (androst-4- derivative forming under storage conditions via a mixed ene-3,17-dione, testosterone, norethisterone, progesterone and anhydride is especially interesting.246 The structure elucidation the 17-OH and 20-OH derivative of the latter). Higher peak of impurities in (among others an efficiencies were obtainable by MEKC and CEC.220 With an in- interesting –S–S– bridged dimer) is a good example for the house packing method of capillaries up to 190.000 plates per application of new techniques such as CEC-MS228 and on-line meter was achieved.221 High-temperature CEC and temperature HPLC-NMR.247 The light-induced degradation of programming resulted in a considerable reduction of the methylprednisolone suleptamate lead to interesting, separation run time of steroids, and the latter may be a good unpredictable structures.248 Of the (contraceptive) gestogens alternative to solvent programming.222 Usually ODS phases are (norethisterone, norgestrel) by-products of their ethinylation used for steroid separations but the successful use of step,240,249 oxidative degradation products of these250 and of macroporous, spherical polystyrene-divinylbenzene stationary nestorone239 are described. Z and E isomers of the 17-(3′- phase was also described.223 Of the practical applications the acetoxy-2′-butenoate)251,252 and 17-(3-oxo-butenoate)1 identified rapid analysis of norgestimate and its degradants,224 in ethynodiol diacetate are by-products of the acetylation of the and related impurities225 and fluticasone propionate and related 17-OH group. Impurities found in allylestrenol originate from impurities226 is mentioned. An example for chiral CEC is its total synthesis.240 presented in the section “Enantiomeric analysis”. Two estrogens are also worth mentioning. In the course of the The very promising possibilities of the new technique CEC- impurity profiling of estradiol HPLC-UV played predominant 778 ANALYTICAL SCIENCES MAY 2004, VOL. 20 role,253 while HPLC-NMR and HPLC-MS were necessary to determine the structures of five most interesting structures (a 8 References hydroperoxide and four different dimers) in the course of oxidative and light-stress studies of ethinylestradiol.254 1. M. Babják, G. Balogh, M. Gazdag, and S. Görög, J. Pharm. Of the other hormonal drugs, several impurities originating Biomed. Anal., 2002, 29, 1153. from the Birch reduction step were identified in 2. S. Görög and Gy. Szász, “Analysis of Steroid Hormone norethisterone,239,250 an isomeric impurity in danazol255 and Drugs”, 1978, Elsevier, Amsterdam. several oxidative degradation products in tipredane.225,256 Of the 3. S. Görög, “Quantitative Analysis of Steroids”, 1983, non-hormonal steroid drugs pipecuronium bromide is Elsevier, Amsterdam. mentioned. An oxidative degradation product243,251 and 4. S. Görög (ed.), “Steroid Analysis in the Pharmaceutical synthesis-related impurities were identified and quantificated by Industry”, 1989, Ellis Horwood, Chichester. NP-HPLC,132 NP-ion-pairing HPLC257 and the oxidative 5. H. L. Makin , D. B. Gower, and D. N. Kirk (ed.), “Steroid degradant also by quantitative NMR spectroscopic Analysis”, 1995, Blackie, Glasgow. measurement.251 6. European Pharmacopoeia 4, Council of , Strasbourg, 2002. 7·2 Enantiomeric analysis 7. United States Pharmacopoeia 26, USP Convention Inc., Although steroid hormone drugs usually contain 6 – 7 chiral Rockville, 2003. centers, their configuration is fixed in natural steroids and the 8. The Japanese Pharmacopoeia 14th Edition, Society of semi-synthetic drugs prepared from them. For this reason Japanese Pharmacopoeia, Tokyo, 2001. enantiomeric analysis is not necessary in the overwhelming 9. J. Bartos and M, Pesez, “Colorimetric and Fluorimetric majority of cases. The only important exception is the total Analysis of Steroids”, 1976, Academic Press, London. synthetically prepared norgestrel, which is mainly administered 10. S. Görög, “Ultraviolet-Visible Spectrophotometry in as enantiomerically pure levonorgestrel, and also (at a Pharmaceutical Analysis”, 1995, CRC Press, Boca Raton, decreasing extent) as the racemate. Several papers have been 323 – 334. published on the separation of the of norgestrel. 11. U. Rose and J. Fuchs, Pharmeuropa, 1998, 10, 146. Only a few of these deal with the determination of the 12. D. Bonazzi, V. Andrisano, R. Gatti, and V. Cavrini, J. enantiomeric purity of levonorgestrel. In the first validated Pharm. Biomed. Anal., 1995, 13, 1321. method RP-HPLC with γ-cyclodextrin in the mobile phase was 13. M. L. Shively and A. P. Simonelli, Int. J. Pharm., 1989, 50, used and a limit of quantitation of 0.1% dextronorgestrel was 39. achieved.258 (With β-cyclodextrin fairly good separation is 14. N. Erk, Anal. Lett., 1999, 32, 1371. 259 achievable at 0˚C only.) Using α1-acid glycoprotein (Chiral 15. M. S. Linares, J. M. G. Fraga, A. I. Jimenez, F. Jimenez, AGP) column for the HPLC separation good separation was and J. J. Arias, Anal. Lett., 1999, 32, 2489. obtained,260,261 but the limit of quantitation was inferior 16. M. L. Luis, J. M. Garcia, A. I. Jimenez, F. Jimenez, and J. (0.6%).260 J. Arias, JAOAC Internat., 1999, 82, 1054. Norgestrel is an excellent model compound for testing the 17. J. J. Berzas, J. Rodríguez Flores, and G. Castañeda, Anal. separation efficiency of various enentioselective Chim. Acta, 1997, 340, 257. chromatographic and related techniques. For this reason many 18. J. J. Berzas, J. Rodríguez, and G. Castañeda, Anal. Sci., papers contain data for norgestrel, usually among many other 1997, 13, 1029. model compounds. Acceptable to excellent separation was 19. J. M. Lemus Gallego and J. Pérez Arroyo, Anal. Chim. achieved using Chiralcel-OJ,262 cellulose tris(3,5- Acta, 2002, 460, 85. dichlorophenylcarbamate),261 amylose tris(3,5-dimethylphenyl- 20. A. J. Nepote, P. C. Damiani, and A .C. Olivieri, J. Pharm. carbamate),263,264 and covalently bonded β-cyclodextrin,265–267 Biomed. Anal., 2003, 31, 621. even bonded to a monolithic silica column.267 SFC using 21. J. M. Lemus Gallego and J. Pérez Arroyo, Anal. Chim. immobilized polysiloxane-anchored permethyl-β-cyclodextrin Acta, 2001, 437, 247. (Chirasil-Dex)268 and teicoplanin or its aglycone (Chirobiotic T 22. H. C. Goicoechea, M. S. Collado, M. L. Satuf, and A. C. or TAG)269 was also used to separate the enantiomers of Olivieri, Anal. Bioanal. Chem., 2002, 374, 460. norgestrel. The above-mentioned cellulose tris(3,5-dichloro- 23. E. R. M. Kedor-Hackmann, E. A. S. Gianottto, and M. I. R. phenylcarbamate) and amylose tris(3,5-dimethylphenyl- M. Santoro, Anal. Lett., 1997, 30, 1861. carbamate) were successfully used for CEC separation of the 24. Y. S. El-Saharty, N. Y. Hassan, and F. H. Metwally, J. enantiomers.270,271 A comparison of the latter in the HPLC and Pharm. Biomed. Anal., 2002, 28, 569. CEC modes revealed significant advantages of CEC.271 This 25. J. J. Berzas, J. Rodríguez, and G. Castañeda, Analyst, 1997, chiral selector was successfully used also for the HPLC 122, 41. separation of as many as 26 steroid enantiomeric pairs including 26. S. Tatar and S. Atmaca, Pharmazie, 1996, 51, 4. estradiol, estrone, ethinylestradiol, nandrolone, etc.263,264 27. J. J. Berzas, J. Rodríguez, G. Castañeda, and F. J. Guzmán 1H-NMR was found to be an excellent tool for modelling Bernardo, Anal. Lett., 1997, 30, 2221. interactions between norgestrel enantiomers and cyclodextrins, 28. T. G. Altuntas, F. Korkmaz, and D. Nebioglu, Pharmazie, thus creating the possibility to find a correlation between the 2000, 55, 1. NMR and RP-HPLC data.272 In addition to this, NMR 29. M. I. Toral, C. Soto, P. Richter, and A. E. Tapia, JAOAC spectroscopy using γ-cyclodextrin273 or the lanthanide shift Internat., 2002, 85, 883. reagent praseodymium tris[3-heptafluoropropyl- 30. E. Dinç, C. Yücesoy, Í. Murat Palabıyık, Ö. Üstündag˘, and hydroxymethylene)-(+)-camphorate]260 was found to be suitable F. Onur, J. Pharm. Biomed. Anal., 2003, 32, 1. for estimating the enantiomeric purity of levonorgestrel without 31. J. M. Lemus Gallego and J. Pérez Arroyo, Microchim. separation. The limit of detection is somewhat higher than that Acta, 2003, 141, 133. achievable by HPLC. 32. J. M. Lemus Gallego and J. Pérez Arroyo, Anal. Lett., 2001, 34, 1265. ANALYTICAL SCIENCES MAY 2004, VOL. 20 779

33. E. Martín, O. Hernández, J. J. Arias, and A. I. Jiménez, 64. H. E. Hauck, A. Junker-Buchheit, and R. Wenig, LC-GC Microchem. J., 1997, 56, 207. Int., 1995, 8, 34. 34. J. Studer, N. Purdie, and J. A. Krouse, Appl. Spectrosc., 65. S. N. Meyyanathan, G. V. S. Ramasarma, and B. Suresh, J. 2003, 57, 791. Planar Chrom., 2001, 14, 188. 35. L. Ayllón, M. Silva, and D. Pérez-Bendito, J. Pharm. Sci., 66. L. Wulandari and G. Idrayanto, J. Liq. Chrom. Rel. 1994, 83, 1135. Technol., 2003, 26, 2709. 36. M. Blanco, J. Coello, H. Iturriaga, S. Maspoch, and N. 67. L. Wulandari, T. K. Sia, and G. Idrayanto, J. Liq. Chrom. Villegas, Analyst, 1999, 124, 911. Rel. Technol., 2003, 26, 109. 37. D. N. Xie, J. H. Ge, Q. T. Lan, and R. G. Yu, Zhongguo 68. J. Novakovic´, I. Neˇmcová, and M. Vasˇtová, J. Planar Yaoke Daxue Xuebao, 1992, 23, 122. Chrom., 1990, 3, 407. 38. C. Vannecke, A. Nguyen Minh Nguyet, M. S. Bloomfield, 69. K. Pavic´, O. Cˇ udina, D. Agbaba, and S. Vladimirov, J. A. J. Staple, Y. Vander Heyden, and D. L. Massart, J. Planar Chrom., 2003, 16, 45. Pharm. Biomed. Anal., 2000, 23, 291. 70. D. Agbaba, Z. Milojevic, S. Eric, M. Aleksic, G. Markovic, 39. E. G. Salole, in “Advances in Steroid Analysis ’90”, ed. S. and M. Solujic, J. Planar Chrom., 2001, 14, 322. Görög, 1991, Akadémiai Kiadó, Budapest, 473. 71. D. Agbaba, S. Eric, G. Markovic, V. Nedeljkovic, S. 40. G. A. Neville, H. D. Beckstead, and J. D. Cooney, Veselinovic, and M. Vucetic, J. Planar Chrom., 2000, 13, Fresenius J. Anal. Chem., 1994, 349, 746. 333. 41. G. A. Neville, H. D. Beckstead, and H. F. Shurvell, J. 72. G. Matysik, J. Toczolowski, and A. Matysik, Pharm. Sci., 1992, 81, 1141. Chromatographia, 1995, 40, 737. 42. H. D. Beckstead, G. A. Neville, and H. F. Shurvell, 73. J. Sherma, B. P. Whitcomb, and K. Brubaker, J. Planar Fresenius J. Anal. Chem., 1993, 345, 727. Chrom., 1990, 3, 189. 43. M. Bartolomei, M. C. Ramusino, and P. Ghetti, J. Pharm. 74. P. N. Kotyiyan and P. R. Vavia, J. Pharm. Biomed. Anal., Biomed. Anal., 1997, 15, 1813. 2000, 22, 667. 44. M. Bartolomei, J. Pharm. Biomed. Anal., 2000, 24, 81. 75. K. Datta and S. K. Das, J. Planar Chrom., 1993, 6, 204. 45. J. A. Ryan, S. V. Compton, M. A. Brooks, and D. A. C. 76. H. Lamparczyk, R. J. Ochoczka, P. Zarzycki, and J. P. Compton, J. Pharm. Biomed. Anal., 1991, 9, 303. Zielin´ski, J. Planar Chrom., 1990, 3, 34. 46. S. Claussen, M. Böse, and M. Dittgen, Pharmazie, 2001, 77. K. Ferenczi-Fodor, S. Mahó, S. Pap-Sziklay, I. Török, and 56, 475. L. Borka, Pharmeuropa, 1997, 9, 736. 47. P. Corti, E. Dreassi, G. Corbini, S. Lonardi, and S. Gravina, 78. A. Wiszkidenszky, S. Mahó, Z. Végh, and K. Ferenczi- Analusis, 1990, 18, 112. Fodor, J. Planar Chrom., 1998, 11, 463. 48. A. M. Belu, M. C. Davies, J. M. Newton, and N. Patel, 79. B. Bagócsi, D. Fábián, A. Laukó, M. Mezei, S. Mahó, Z. Anal. Chem., 2000, 72, 5625. Végh, and K. Ferenczi-Fodor, J. Planar Chrom., 2002, 15, 49. A. Lommen, R. Schilt, J. Weseman, A. H. Roos, J. W. van 252. Velde, and M. W. F. Nielen, J. Pharm. Biomed. Anal., 80. B. Bagócsi, G. Rippel, M. Mezei, Z. Végh, and K. 2002, 28, 87. Ferenczi-Fodor, J. Planar Chrom., 2003, 16, 359. 50. P. J. Saindon, N. S. Cauchon, P. A. Sutton, C.-J. Chang, G. 81. A. Kassai, A. Szécsi, A. Koppány, Z. Végh, and K. E. Peck, and S. R. Byrn, Pharm. Res., 1993, 10, 197. Ferenczi-Fodor, J. Planar Chrom., 2000, 13, 30. 51. U. Skogsberg, H. Händel, E. Gesele, T. Sokolliess, U. 82. Z. Katona, L. Vincze, Z. Végh, Á. Trompler, and K. Menyes, T. Jira, U. Roth, and K. Albert, J. Sep. Sci., 2003, Ferenczi-Fodor, J. Pharm. Biomed. Anal., 2000, 22, 252. 26, 1119. 83. P. K. Zarzycki, J. Chromatogr. A, 2002, 971, 193. 52. I. I. Koukli and A. C. Calokerinos, Analyst, 1990, 115, 84. M. Matyska, A.-M. Siouffi, and E. Soczewinski, J. Planar 1553. Chrom., 1991, 4, 255. 53. N. T. Deftereos and A. C. Calokerinos, Anal. Chim. Acta, 85. H. E. Hauck, O. Bund, W. Fischer, and M. Schulz, J. 1994, 290, 190. Planar Chrom., 2001, 14, 234. 54. Y. Iglesias, C. Fente, B. I Vazquez, C. Franco, A. Cepeda, 86. K. Ferenczi-Fodor, A. Laukó, A. Wiszkidenszky, Z. Végh, and S. Mayo, Anal. Chim. Acta, 2002, 468, 43. and K. Újszászy, J. Planar Chrom., 1999, 12, 30. 55. A. Gergely, in “Analytical Applications of Circular 87. L. Gagliardi, D. de Orsi, M. R. del Giudice, F. Gatta, R. Dichroism”, ed. N. Purdie and H. G Brittain, 1994, Porrà, P. Chimenti, and D. Tonelli, Anal. Chim. Acta, 2002, Elsevier, Amsterdam, 293. 457, 187. 56. D. Szegvári, P. Horváth, A. Gergely, S. Németh, and S. 88. J. Krzek, U. Hubicka, M. Dabrowska-Tylka, and E. Görög, Anal. Bioanal. Chem., 2003, 375, 713. Leciejewicz-Ziemecka, Chromatographia, 2002, 56, 759. 57. A. Szentesi, A. Gergely, P. Horváth, and Gy. Szász, 89. E. Olszewska, W. Kroszczynski, M. Lypacewicz, and T. Fresenius’ J. Anal. Chem., 2000, 368, 384. Wasiak, Acta Chrom., 2002, 12, 219. 58. G. Indrayanto, L. Aditama, W. Tanudjaja, and S. Widjaja, 90. M. Waksmundzka-Hajnos, A. Petrucznik, and A. Hawryl, J. Planar Chrom., 1998, 11, 201. Chem. Anal., 2003, 48, 453. 59. G. Indrayanto, I. Wahyuningsih, and R. J. Salim, J. Planar 91. M. Waksmundzka-Hajnos, M. L. Bieganowska, and A. Chrom., 1997, 10, 204. Petrucznik, J. Planar Chrom., 1992, 5, 446. 60. G. Indrayanto, S. Widjaja, and S. Sutiono, J. Liq. 92. N. A. Zakhari, M. I. Walash, M. S. Rizk, S. S. Toubar, C. J. Chromatogr. Relat. Technol., 1999, 22, 143. W. Brooks, and W. J. Cole, J. Pharm. Biomed. Anal., 1991, 61. J. Novakovic´, D. Agbaba, S. Vladimirov, and D. Zˇ ivanov- 9, 705. Stakic´, J. Pharm. Biomed. Anal., 1990, 8, 253. 93. J. Novakovic, E. Tvrzicka, and A. Pisarikova, Anal. Lett., 62. K. Ferenczi-Fodor, Z. Végh, and Z. Pap-Sziklay, J. Planar 1991, 24, 1559. Chrom., 1993, 6, 198. 94. R. Hsu and A. M. Au, Bull. Environ. Contam. Toxicol., 63. K. Ferenczi-Fodor, A. Nagy-Turák, and Z. Végh, J. Planar 2001, 66, 178. Chrom., 1995, 8, 349. 95. A. B. Fialkov, A. Gordin, and A. Amirav, J. Chromatogr. 780 ANALYTICAL SCIENCES MAY 2004, VOL. 20

A, 2003, 991, 217. 128.V. Pucci, F. Bugamelli, R. Mandrioli, B. Luppi, and M. A. 96. K. J. S. De Cock, F. T. Delbeke, P. Van Eenoo, N. Desmet, Raggi, J. Pharm. Biomed. Anal., 2003, 30, 1549. K. Roels, and P. De Backer, J. Pharm. Biomed. Anal., 129.R. Gonzalo-Lumbreras, A. Santos-Montes, E. Garcia- 2001, 25, 843. Moreno, and R. Izquierdo-Hornillos, J. Chromatogr. Sci., 97. F. T. Delbeke, P. Van Eenoo, W. Van Thuyne, and N. 1997, 35, 439. Desmet, J. Steroid Biochem. Mol. Biol., 2003, 83, 245. 130.P. A. D. Edwardson and R. S. Gardner, J. Pharm. Biomed. 98. Alltech Application Note, 0047E, May 2000, 2. Anal., 1990, 8, 935. 99. J. S. Loran and K. D. Cromie, J. Pharm. Biomed. Anal., 131.L. Matysová, R. Hájková, J. Sˇicha, and P. Solich, Anal. 1990, 7, 607. Bioanal. Chem., 2003, 376, 440. 100.J. R. Dean and J. Lowdon, Analyst, 1993, 118, 747. 132.G. Szepesi, M. Gazdag, and K. Mihályfi, J. Chromatogr., 101.Y. Yamini, M. Asghari-Khiavi, and N. Bahramifar, 1989, 464, 265. Talanta, 2002, 58, 1003. 133.S. Hou, M. Hindle, and P. R. Byron, J. Pharm. Biomed. 102.L. Zivanovic, L. Vojvodic, P. Ristic, M. Zecevic, and I. Anal., 2001, 24, 371. Nemcova, Biomed. Chromatogr., 2000, 14, 56. 134.A. M. Kaukonen, P. Vuorela, H. Vuorela, and J.-P. 103.M. LeBelle, R. K. Pike, S. J. Graham, E. D. Ormsby, and Mannermaa, J. Chromatogr., A, 1998, 797, 271. H. A. Bogard, J. Pharm. Biomed. Anal., 1996, 14, 793. 135.E. A. Gad Kariem, M. A. Abounassif, M. E. Hagga, and H. 104.D. De Orsi, L. Gagliardi, F. Chimenti, and D. Tonelli, Anal. A. Al-Khamees, J. Pharm. Biomed. Anal., 2000, 23, 413. Lett., 1995, 28, 1655. 136.R. Gonzalo-Lumbreras and R. Izquierdo-Hornillos, J. 105.J. E. Kountourellis, C. K. Markopoulou, K. O. Ebete, and J. Chromatogr. B, 2000, 742, 1. A. Stratis, J. Liq. Chromatogr., 1995, 18, 3507. 137.J. C. Reepmeyer, J. Liq. Chromatogr. Relat. Technol., 106.R. D. Marini, A. Pantella, M. A. Bimazubute, P. Chiap, P. 2001, 693. Hubert, and J. Crommen, Chromatographia, 2002, 55, 263. 138.M. Herzler, S. Herre, and F. Pragst, J. Anal. Toxicol., 2003, 107.K.-R. Liu, S.-H. Chen, S.-M. Wu, H.-S. Kou, and H.-L. 27, 233. Wu, J. Chromatogr. A, 1994, 676, 455. 139.L. Valvo, A. Paris, A. L. Savella, B. Gallinella, and E. 108.A. Santos Montes, A. I. Gasco Lopez, and R. Izquierdo Ciranni Signoretti, J. Pharm. Biomed. Anal., 1994, 12, 805. Hornillos, Chromatographia, 1994, 39, 539. 140.M. W. F. Nielen, J. P. C. Vissers, R. E. M. Fuchs, J. W. van 109.L. González, G. Yuln, and M. G. Volonté, J. Pharm. Velde, and A. Lommen, Rapid Commun. Mass Spectrom., Biomed. Anal., 1999, 20, 487. 2001, 15, 1577. 110.A. Segall, M. Vitale, V. Pérez, F. Hormaechea, M. 141.D. Hooijerink, R. Schilt, E. van Bennekom, and B. Palacios, and M. T. Pizzorno, Drug Dev. Int. Pharm., 2000, Brouwer, Analyst, 1994, 119, 2617. 26, 867. 142.K. Yu and M. Balogh, LC-GC North America, 2001, 19, 60. 111.Z. Milojevic, D. Agbaba, S. Eric, D. Boberic-Borojevic, P. 143.S. Brombacher, S. J. Owen, and D. A. Volmer, Anal. Ristic, and M. Solujic, J. Chromatogr. A, 2002, 949, 79. Bioanal. Chem., 2003, 376, 773. 112.J. P. Surmann, B. Strahl, and C. Hock, Pharmazie, 1994, 144.J. Lindholm, D. Westerlund, K.-E. Karlsson, K. Caldwell, 49, 139. and T. Forstedt, J. Chromatogr. A, 2003, 992, 85. 113.J. M. Lemus Gallego and J. Pérez Arroyo, J. Pharm. 145.J. Lindholm, M. Johansson, and T. Forstedt, J. Biomed. Anal., 2002, 30, 1255. Chromatogr. B, 2003, 791, 323. 114.M. I. R. M. Santoro, E. B. Govato, and E. R. M. 146.T. Glaser and K. Albert, J. Sep. Sci., 2002, 25, 393. Hackmann, Anal. Lett., 1993, 26, 925. 147.K. Kofuji, T. Ito, Y. Murata, and S. Kawashima, Biol. 115.J. Li, J. Pharm. Sci., 2002, 91, 1873. Pharm. Bull., 2001, 24, 205; Biol. Pharm. Bull., 2002, 25, 116.A. Segall, F. Hormaechea, M. Vitale, V. Pérez, and M. T. 268. Pizzorno, J. Pharm. Biomed. Anal., 1999, 19, 803. 148.C. C. Leffler and B. W. Müller, S. T. P. Pharma Sci., 2000, 117.M. I. R. M. Santoro, N. M. Kassab, M. Hasegawa, and E. 10, 105. R. M. Kedor-Hackmann, Drug Dev. Ind. Pharm., 2002, 28, 149.J. A. Russel, R. K. Malcolm, K. Campbell, and A. D. 741. Woolfson, J. Chromatogr. B, 2000, 744, 157. 118.A. A. Syed and M. K. Amshumali, J. Pharm. Biomed. 150.P. R. Rege, V. D. Vivivalam, and C. C. Collins, J. Pharm. Anal., 2001, 25, 1015. Biomed. Anal., 1998, 17, 1225. 119.O. Cudina, J. Brboric, Z. Vujic, D. Radulovic, and S. 151.J. K. Lim, M. C. Riley, and S. T. Watts, LC-GC North Vladimirov, Farmaco, 2000, 55, 125. America, 2003, 21, 47. 120.J. M. Lemus Gallego and J. Pérez Arroyo, 152.D. Sˇatinski, J. Hulcová, P. Solich, and R. Karlicˇek, J. Chromatographia, 2002, 55, 749. Chromatogr. A, 2003, 1015, 239. 121.J. M. Lemus Gallego and J. Pérez Arroyo, Anal. Bioanal. 153.L. A. Romanyshyn and P. R. Tiller, J. Chromatogr. A, Chem., 2002, 374, 282. 2001, 928, 41. 122.R. Hájková, P. Solich, J. Dvorˇak, and J. Sˇicha, J. Pharm. 154.H. Kanazawa, K. Yamamoto, Y. Matsushima, N. Takai, A. Biomed. Anal., 2003, 32, 921. Kikuchi, Y. Sakurai, and T. Okano, Anal. Chem., 1996, 68, 123.M. J. Galmier, E. Beyssac, J. Petit, J. M. Aiache, and C. 105. Lartigue, J. Pharm. Biomed. Anal., 1999, 20, 405. 155.H. Kanazawa, T. Sunamoto, E. Ayano, Y. Matsushima, A. 124.S. M. Ahmed, F. Arcuri, F. Li, A. J. Moo-Young, and C. Kikuchi, and T. Okano, Anal. Sci., 2002, 18, 45. Monder, Steroids, 1995, 60, 534. 156.Y. X. Song, J. Q. Wang, Z. X. Su, and D. Y. Chen, 125.M. Zecevic, Lj. Zivanovic, and A. Stojkovic, J. Chromatographia, 2001, 54, 208. Chromatogr., 2002, 949, 61. 157.B. Buszewski, M. Jezierska-S´witala, and S. Kowalska, J. 126.M. S. Ali, M. Ghori, and A. Saeed, J. Chromatogr. Sci., Chromatogr. B, 2003, 792, 279. 2002, 40, 429. 158.C. Baggiani, G. Giraudi, F. Trotta, C. Giovannoli, and A. 127.W. J. Bachman and J. G. Gambertoglio, Anal. Lett., 1990, Vanni, Talanta, 2000, 51, 71. 23, 893. 159.L. Ye, Y. Yu, and K. Mosbach, Analyst, 2001, 126, 760. ANALYTICAL SCIENCES MAY 2004, VOL. 20 781

160.E.-Y. Ting and M. D. Porter, Anal. Chem., 1997, 69, 675. 381. 161.H. Deng, G. J. Van Berkel, H. Takano, D. Gazda, and M. 191.X.-Y. Liu, C. Nakamura, Q. Yang, N. Kamo, and J. D. Porter, Anal. Chem., 2000, 72, 2641. Miyake, J. Chromatogr. A, 2002, 961, 113. 162.J. L. Bernal, M. J. Del Nozal, G. A. Garcia Buj, and J. M. 192.Y. Zhao, J. Jona, D. T. Chow, H. Rong, D. Semin, X. Xia, Juárez, J. Chromatogr., 1992, 607, 175. R. Zanon, C. Spancake, and E. Maliski, Rapid Commun. 163.K. Valkó, S. Espinosa, C. M. Du, E. Bosch, M. Rosés, C. Mass Spectrom., 2002, 16, 1548. Bevan, and M. H. Abraham, J. Chromatogr. A, 2001, 933, 193.M. Molero-Monfort, L. Escuder-Gilabert, R. M. 73. Villanueva-Camañas, S. Sagrado, and M. J. Medina- 164.M. Z. Kagan, J. Chromatogr., 2001, 918, 293. Hernández, J. Chromatogr. B, 2001, 753, 225. 165.P. K. Zarzycki, K. M. Kulhanek, and R. Smith, J. 194.J. Li, J. Chromatogr. A, 2001, 927, 19. Chromatogr. A, 2002, 955, 71. 195.J. Li and D. S. Shah, J. Chromatogr. A, 2002, 954, 159. 166.P. K. Zarzycki and R. Smith, J. Chromatogr. A, 2001, 912, 196.J. M. Lemus Gallego and J. Pérez Arroyo, Anal. Lett., 45. 2002, 35, 2105. 167.A. Pyka, J. Liq. Chromatogr. Relat. Technol., 2001, 24, 197.S. Görög, M. Gazdag, and P. Kemenes-Bakos, J. Pharm. 453. Biomed. Anal., 1996, 14, 1115. 168.E. Bonet-Domingo, M. J. Medina-Hernandez, and M. C. 198.P. Britz-McKibbin, T. Ichihashi, K. Tsubota, D. D. Y. Garcia-Alvarez-Coque, J. Pharm. Biomed. Anal., 1993, 8, Chen, and S. Terabe, J. Chromatogr. A, 2003, 1013, 65. 711. 199.H. Nishi, T. Fukuyama, M. Matsuo, and S. Terabe, J. 169.S. Torres-Cartas, R. M. Villanueva-Camañas, and M. C. Chromatogr., 1990, 513, 279. Garcia-Alvarez-Coque, Chromatographia, 2000, 51, 577. 200.S. Terabe, Y. Ishihama, H. Nishi, T. Fukuyama, and K. 170.S. Torres-Cartas, J. R. Torres-Lapasio, R. M. Villanueva- Otsuka, J. Chromatogr., 1991, 545, 359. Camañas, and M. C. Garcia-Alvarez-Coque, 201.J. Vindevogel and P. Sandra, Anal. Chem., 1991, 63, 1530. Chromatographia, 2000, 52, 185. 202.M. Lin and N. Wu, J. Pharm. Biomed. Anal., 1999, 19, 945. 171.S. Torres-Cartas, R. M. Villanueva-Camañas, and M .C. 203.J. J. Berzas, B. Del Castillo, G. Castañeda, and M. J. Garcia-Alvarez-Coque, J. Liq. Chromatogr. Relat. Pinilla, Talanta, 1999, 50, 261. Technol., 2000, 23, 1171. 204.J. J. Berzas, J. Rodríguez, G. Castañeda, and M. J. Pinilla, 172.M. J. Ruiz-Angel, R. D. Caballero, E. F. Simó-Alfonso, and Chromatographia, 1999, 49, 65. M. C. Garcia-Alvarez-Coque, J. Chromatogr., 2002, 947, 205.J. M. Lemus Gallego and J. Pérez Arroyo, J. Liq. 31. Chromatogr. Relat. Technol., 2003, 26, 1011. 173.M.-E. Capella-Peiró, M. Gil-Augusti, L. Monferrer-Pons, 206.J. M. Lemus Gallego and J. Pérez Arroyo, and J. Esteve-Romero, Anal. Chim. Acta, 2002, 454, 125. Chromatographia, 2003, 58, 277. 174.R. Gonzalo-Lumbreras and R. Izquierdo-Hornillos, J. 207.J. M. Lemus Gallego and J. Pérez Arroyo, J. Pharm. Pharm. Biomed. Anal., 2003, 31, 201. Biomed. Anal., 2003, 31, 873. 175.R. Gonzalo-Lumbreras and R. Izquierdo-Hornillos, J. 208.J. M. Lemus Gallego and J. Pérez Arroyo, Fresenius’ J. Pharm. Biomed. Anal., 2003, 32, 433. Anal. Chem., 2001, 370, 973. 176.R. Gonzalo-Lumbreras and R. Izquierdo-Hornillos, J. 209.S. E. Lucangioli, V. G. Rodríguez, G. C. Fernandez Otero, Chromatogr., 2003, 794, 215. N. M. Vizioli, and C. N. Carducci, J. Capillary 177.N. S. Wilson, M. D. Nelson, J. W. Dolan, L. R. Snyder, R. Electrophor., 1997, 4, 27. G. Wolcott, and P. W. Carr, J. Chromatogr. A, 2002, 961, 210.S. Noé, J. Böhler, E. Keller, and A. W. Frahm, J. Pharm. 171, 195. Biomed. Anal., 1998, 18, 911. 178.V. Morris, J. Hughes, and P. Marriott, J. Chromatogr. A, 211.A. Cifuentes, J. L. Bernal, and J. C. Diez-Masa, J. 2003, 1008, 43. Chromatogr. A, 1998, 824, 99. 179.D. Barron, J. A. Pascual, J. Segura, and J. Barbosa, 212.H. Okamoto, A. Uetake, R. Tamaya, T. Nakajima, K. Chromatographia, 1995, 41, 573. Sagara, and Y. Ito, J. Chromatogr. A, 2001, 929, 133. 180.Y. S. Gau, S. W. Sun, and R.-L. Chen, J. Liq. Chromatogr., 213.M. R. N. Monton, K. Otsuka, and S. Terabe, J. 1995, 18, 2373. Chromatogr. A, 2003, 985, 435. 181.I. Cenderowska and B. Buszewski, J. Liq. Chromatogr. 214.L. Vomastová, I. Miksˇik, and Z. Deyl, J. Chromatogr. B, Relat. Technol., 1999, 22, 2259. 1996, 681, 107. 182.V. Das Gupta and M. Mathew, Drug Dev. Ind. Pharm., 215.S. Pedersen-Bjergaard, C. Gabel-Jensen, and S. H. Hansen, 1995, 21, 833. J. Chromatogr. A, 2000, 897, 375. 183.J. Zhu and C. Coscolluella, J. Chromatogr. B, 2000, 741, 216.S. H. Hansen, C. Gabel-Jensen, and S. Pedersen-Bjergaard, 55. J. Sep. Sci., 2001, 24, 643. 184.N. H. Anderson, M. R. Gray, and C. J. Hinds, J. Pharm. 217.C. Gabel-Jensen, S. H. Hansen, and S. Pedersen-Bjergaard, Biomed. Anal., 1990, 8, 853. Electrophoresis, 2001, 22, 1330. 185.M. Mulholland, T. J. Whelan, H. Rose, and J. Keegan, J. 218.S. E. Lucangioli, C. N. Carducci, S. L. Scioscia, A. Chromatogr. A, 2000, 870, 135. Carlucci, C. Bregni, and E. Kenndler, Electrophoresis, 186.W. J. Bachman and J. T. Stewart, J. Chromatogr. Sci., 2003, 24, 984. 1990, 28, 123. 219.S. K. Poole, D. Durham, and C. Kibbey, J. Chromatogr., 187.A. M. Di Pietra, V. Andrisano, R. Gotti, and V. Cavrini, J. 2000, 745, 117; 2003, 793, 265. Pharm. Biomed. Anal., 1996, 14, 1191. 220.R. B. Taylor, S. Vorarat, R. G. Reid, S. P. Boyle, and R. R. 188.Alltech Application Note, 0046E, 2000, 2. Moody, J. Capillary Electrophor., 1999, 6, 131. 189.K. G. Flood, E. R. Reynolds, and N. H. Snow, J. 221.J. Saevels, M. Wuyts, A. Van Schepdael, E. Roets, and J. Chromatogr. A, 2001, 913, 261. Hoogmartens, J. Pharm. Biomed. Anal., 1999, 20, 513. 190.E. H. Kerns, L. Di, S. Petusky, T. Kleintop, D. Huryn, O. 222.N. M. Djordjevic, F. Fitzpatrick, F. Houdiere, G. Lerch, McConnel, and G. Carter, J. Chromatogr. B, 2003, 791, and G. Rozing, J. Chromatogr., 2000, 887, 245. 782 ANALYTICAL SCIENCES MAY 2004, VOL. 20

223.Y. Liu and D. J. Pietrzyk, J. Chromatogr. A, 2001, 920, 249.P. Horváth, G. Balogh, J. Brlik, A, Csehi, F. Dravecz, Zs. 367. Halmos, A. Laukó, M. Rényei, K. Varga, and S. Görög, J. 224.J. Wang, D. E. Schaufelberger, and N. A. Guzman, J. Pharm. Biomed. Anal., 1997, 15, 1343. Chromatogr. Sci., 1998, 36, 155. 250.S. Görög, M. Bihari, É. Csizér, F. Dravecz, M. Gazdag, and 225.M. R. Euerby, D. Gilligan, C. M. Johnson, S. C. P. Roulin, B. Herényi, J. Pharm. Biomed. Anal., 1995, 14, 85. P. Myers, and K. D. Bartle, J. Microcol. Sep., 1997, 9, 373. 251.S. Görög, G. Balogh, and M. Gazdag, J. Pharm. Biomed. 226.N. W. Smith and M. B. Evans, Chromatographia, 1994, 38, Anal., 1991, 9, 829. 649; 1995, 41, 197. 252.S. Görög, Zs. Halmos, B. Herényi, A. Georgakis, G. 227.S. J. Lane, in “Identification and Determination of Balogh, É. Csizér, and Z. Tuba, in “Advances in Steroid Impurities in Drugs”, ed. S. Görög, 2000, Elsevier, Analysis ’90”, ed. S. Görög, 1991, Akadémiai Kiadó, Amsterdam, 359 – 381. Budapest, 323 – 329. 228.S. J. Lane, R. Boughtflower, C. Paterson, and T. 253.S. Görög, J. Brlik, A, Csehi, Zs. Halmos, B. Herényi, P. Underwood, Rapid Commun. Mass Spectrom., 1995, 9, Horváth, F. Dravecz, and D. Bor, Anal. Meth. Instrum., 1283. 1995, 2, 154. 229.R. N. Warriner, A. S. Craze, D. E. Games, and S. J. Lane, 254.B. E. Segmuller, B. L. Armstrong, R. Dunphy, and A. R. Rapid Commun. Mass Spectrom., 1998, 12, 1143. Oyer, J. Pharm. Biomed. Anal., 2000, 23, 927. 230.P. M. Bersier and J. Bersier, in “Steroid Analysis in the 255.G. Balogh, É. Csizér, Gy. G. Ferenczy, Zs. Halmos, B. Pharmaceutical Industry”, ed. S. Görög, Ellis Horwood, Herényi, P. Horváth, A. Laukó, and S. Görög, Pharm. Res., Chichester, 166 – 180. 1995, 12, 295. 231.F. Belal, Microchim. Acta, 1992, 107, 11. 256.M. R. Euerby, J. A. Graham, C. M. Johnson, R. J. Lewis, 232.M. I. Walash, F. Belal, M. E.-S. Metwally, and M. and D. B. Wallace, J. Pharm. Biomed. Anal., 1996, 15, 299. Hefnawy, Microchim. Acta, 1994, 112, 217. 257.M. Gazdag, M. Babják, P. Kemenes-Bakos, and S. Görög, 233.C. Jesaseelan and A. P. Joshi, Anal. Bioanal. Chem., 2002, J. Chromatogr., 1991, 550, 639. 373, 772. 258.M. Gazdag, G. Szepesi, and K. Mihályfi, J. Chromatogr., 234.J.-E. Belgaied, Anal. Bioanal. Chem., 2003, 376, 706. 1988, 450, 145. 235.J. J. Berzas, J. Rodríguez, and G. Castañeda, 259.H. Lamparczyk, P. K. Zarzycki, and J. Nowakowska, J. Electroanalysis, 1999, 11, 268. Chromatogr. A, 1994, 668, 413. 236.R. Aubeck, C. Bräuchle, and N. Hampp, Anal. Chim. Acta, 260.Y. Blom, M. Ek, J. T. Martin, and N. E. Stjernström, 1990, 238, 405. Pharmeuropa, 1993, 5, 381. 237.S. Görög, “Identification and Determination of Impurities 261.Gy. Szász, K. Gyimesi-Forrás, and Zs. Budvári-Bárány, J. in Drugs”, 2000, Elsevier, Amsterdam, 712 – 731. Liq. Chromatogr. Relat. Technol., 1998, 21, 2535. 238.S. Görög, Anal. Bioanal. Chem., 2003, 377, 852. 262.B. Chankvetadze, I. Kartozia, C. Yamamoto, and Y. 239.S. Görög, M. Babják, G. Balogh, J. Brlik, F. Dravecz, M. Okamoto, J. Pharm. Biomed. Anal., 2002, 27, 467. Gazdag, P. Horváth, A. Laukó, and K. Varga, J. Pharm. 263.M. Kummer, H.-J. Palme, and G. Werner, J. Chromatogr. Biomed. Anal., 1998, 18, 511. A, 1996, 749, 61. 240.S. Görög, G. Balogh, A. Csehi, É. Csizér, M. Gazdag, Zs. 264.M. Kummer and G. Werner, J. Chromatogr. A, 1998, 825, Halmos, B. Hegedüs, B. Herényi, P. Horváth, and A. 107. Laukó, J. Pharm. Biomed. Anal., 1993, 11, 1219. 265.A. Berthod, H. L. Jin, T. E. Beesly, J. D. Duncan, and D. 241.S. Görög, M. Babják, G. Balogh, J. Brlik, A, Csehi, F. W. Armstrong, J. Pharm. Biomed. Anal., 1990, 8, 123. Dravecz, M. Gazdag, P. Horváth, A. Laukó, and K. Varga, 266.Macherey-Nagel Application Note, A-1103, 2001, 2. Talanta, 1997, 44, 1517. 267.D. Lubda, K. Cabrera, K. Nakanishi, and W. Lindner, Anal. 242.M. Gazdag, M. Babják, J. Brlik, S. Mahó, Z. Tuba, and S. Bioanal. Chem., 2003, 377, 892. Görög, J. Pharm. Biomed. Anal., 1998, 17, 1029. 268.M. Jung and V. Schurig, J. High Res. Chrom., 1993, 16, 243.S. Görög, Trends Anal. Chem., 2003, 22, 407. 215. 244.G. Cavina, R. Alimenti, B. Gallinella, and L. Valvo, J. 269.Y. Liu, A. Berthod, C. R. Mitchell, T. L. Xiao, B. Zhang, Pharm. Biomed. Anal., 1992, 9, 685. and D. W. Armstrong, J. Chromatogr. A, 2002, 978, 185. 245.M. Spangler and E. Mularz, Chromatographia, 2001, 54, 270.B. Chankvetadze, I. Kartozia, J. Breitkreutz, Y. Okamoto, 329. and G. Blaschke, Electrophoresis, 2001, 22, 3327. 246.R. E. Conrow, G. W. Dillow, L. Bian, L. Xue, O. 271.L. Chakvetadze, I. Kartozia, C. Yamamoto, B. Papadopoulou, J. K. Baker, and B. S. Scott, J. Org. Chem., Chankvetadze, G. Blaschke, and Y. Okamoto, J. Sep. Sci., 2002, 6835. 2002, 25, 653. 247.N. Mistry, I. M. Ismail, M. G. Smith, J. K. Nicholson, and 272.G. Tárkányi, J. Chromatogr. A, 2002, 961, 257. J. C. Lindon, J. Pharm. Biomed. Anal., 1997, 16, 697. 273.G. Tárkányi, in “Identification and Determination of 248.M. Ogata, Y. Noro, M. Yamada, T. Tahara, and T. Impurities in Drugs”, ed. S. Görög, 2000, Elsevier, Nishimura, J. Pharm. Sci., 1998, 87, 91. Amsterdam, 562 – 574.

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